Suspected insurgents fired on an Afghan government delegation on Tuesday investigating the massacre of 16 civilians by a U.S. soldier, officials said, hours after the Taliban threatened to behead American troops to avenge the killings.

One Afghan soldier was killed in the attack and two others were wounded.

Two of President Hamid Karzai's brothers, Shah Wali Karzai and Addul Qayum Karzai, were with senior defence, intelligence and interior ministry officials travelling to the scene of the massacre in Najiban and Alekozai villages, in Kandahar's Panjwai district, when insurgents opened fire.

Karzai's brothers were unharmed in the brief gunbattle during meetings at a village mosque. The area is a Taliban stronghold and a supply route.

"The Islamic Emirate once again warns the American animals that the mujahideen will avenge them, and with the help of Allah will kill and behead your sadistic murderous soldiers," Taliban spokesman Zabihullah Mujahid said in a statement, using the term with which the Islamist group describes itself.

As the first protest broke out in Jalalabad city over the weekend shootings, the Taliban said Afghan government demands for an open trial of the U.S. Army staff sergeant being held for the slayings would not blunt civilian hostility towards Western combat troops.

The unnamed U.S. soldier – said to have only recently arrived in the country – is accused of walking off his base in Kandahar province in the middle of the night and gunning down at least 16 villagers, mostly women and children.

A U.S. official said the accused soldier had suffered a traumatic brain injury while on a previous deployment in Iraq.

The shootings, which came just weeks after deadly protests across the country over the inadvertent burning of Korans by U.S. soldiers, triggered a protest by around 2,000 students in the eastern city of Jalalabad.

The demonstrators chanted ‘Death to America’ and demanded Afghan President Hamid Karzai reject plans to sign a strategic pact with Washington that would allow U.S. advisers and possibly special forces to remain in the country beyond the planned withdrawal in 2014.

U.S. President Barack Obama, speaking after a phone call with Karzai - who is said to be furious over the latest deaths - said the shootings had only increased his determination to get American troops out of Afghanistan.

However, Obama cautioned there should not be a ‘rush to the exits’ for U.S. forces who have been fighting in Afghanistan since late 2001 and that the drawdown set for the end of 2014 should be done in a responsible way.

The soldier, from a conventional unit, was based at a joint U.S.-Afghan base used by elite U.S. troops under a so-called village support programme hailed by NATO as a possible model for U.S. involvement in the country after the 2014 drawdown.

Such bases provide support to local Afghan security units and provide a source of security advice and training, as well as anti-insurgent backup and intelligence.

Can no longer be called rogue

A spokesman for Kandahar governor Tooryalai Wisa said that tribal elders in the area of the massacre would urge against protests and work to dampen public anger if the investigation process was transparent.

"They are supporting the government and will accept any conclusion by the investigators. Today we have meetings with people in the area and all will become clear," spokesman Ahmad Jawid Faisal said.

NATO officials said it was too early to tell if the U.S. soldier would be tried in the United States or Afghanistan if investigators were to find enough evidence to charge him, but he would be under U.S. laws and procedures under an agreement between U.S. and Afghan officials.

Typically, once the initial investigation is completed, prosecutors decide if they have enough evidence to file charges and then could move to an Article 32 or court martial hearing.

U.S. Defence Secretary Leon Panetta said on Monday that the death penalty could be sought in the U.S. military justice system against the soldier, but portrayed the shooting as an isolated event that would not alter withdrawal plans.

While Afghan MPs in parliament called for a trial under Afghan law, Karzai's office was understood to accept that a trial in a U.S. court would be acceptable provided the process was transparent and open to media.

Analysts said the incident would complicate U.S. efforts to reach agreement with the Afghan government on a post-2014 security pact before a May summit in the U.S. city of Chicago on the future size and funding of Afghan security forces.

Thomas Ruttig of the Afghanistan Analysts Network said that despite NATO and White House references to the killings as the work of a ‘rogue’ soldier, other similar events had happened before, including a ‘kill team’ apprehended in Kandahar in 2010.

"In the stress of an environment of escalated violence - by both sides, but particularly after Obama's troop surge in early 2009, it looks as if most soldiers simply see Afghanistan as a whole as 'enemy territory' and every Afghan as a potential terrorist. This can no longer be called 'rogue'," Ruttig said.

NATO's top commander in Afghanistan, Marine General John Allen, has promised a rapid investigation of the massacre, while security was being reviewed at NATO bases across the country.