The Danish Committee for Aid to Afghan Refugees (DACAAR) sent five employees to Ghazni province on Tuesday. They were attacked by armed men.
The ambush occurred on a road in the district of Ab Band, about 150km south of the capital Kabul.
The unidentified assailants pulled the passengers out of the car before shooting them, but the fifth passenger escaped with injuries.
When contacted at the head office in Copenhagen by Aljazeera.net, DACAAR said no statement would be made until all relatives had been told the tragic news.
The increasing instability is making a huge aid task almost impossible.
“Aid agencies in Afghanistan are understandably shell-shocked by the murder," said United Nations Humanitarian Commission for Refugees spokeswoman Maki Shinohara in Kabul.
Refering to UNHCR workers who have also been killed in recent months, Shinohara added: "Our movements are restricted and it will take some time for staff to regain confidence to work fully in the field and help people in communities."
More than 10 international non-governmental organisations have pulled out of Kandahar, at least on a temporary basis.
UNHCR's Spin Boldak field unit has been closed since the start of the war in Iraq on 20 March. Operations at the Chaman border have also been restricted or stopped.
"Aid agencies in Afghanistan are understandably shell-shocked by the murder"
Kabul office UNHCR
No go zones
Local Afghan officials have also been targeted, and large areas of south-eastern Afghanistan - including the whole of Uruzgan and Zabul provinces - remain off-limits to aid agency staff because of the dangers.
Other areas can only be accessed using armed escorts.
The surge in violence against aid agencies and groups cooperating with the government or US forces in Afghanistan has been blamed on resurgent Taliban fighters, alleged to be regrouping over the border in Pakistan.
The latest attack at Ab Band lies near the southeast border province of Paktika, where resurgent Taliban and suspected al-Qaida have regrouped.
The whole area has witnessed huge military deployments in recent weeks, comprising US special forces and the Afghan Militia Force – at one point commanders were optimistic of capturing Mulla Umar.
Paktika, neighbouring Zabul province and Ghazni are believed to be among the main routes of infiltration used by bands of Taliban from Pakistan.
About 11,500 troops, most of them American, are hunting down Taliban and al-Qaida fighters in Afghanistan.
Anniversay delays security
The attack came on the anniversary of Ahmad Shah Masud’s assassination - an occasion which has delayed Afghan officials from attending talks over the country's volatile border with Pakistan.
Ahmad Shah Masud was a hero
for many Afghans
The fifth round of talks were supposed to discuss Taliban infiltrations on Wednesday, in addition to demarcation disputes along the 2450km mountainous border.
However, US and Pakistani officials have been forced to postpone the meeting after Kabul representatives failed to turn up “because of certain commitments back home”.
Military spokesman Major General Shawkat Sultan told journalists the delay “was because of the death anniversary of Ahmad Shah Masud”.
On Tuesday, interim President Hamid Karzai led a state-organised ceremony in memory of the regional commander.
Issues for discussion
Afghan officials claim Taliban remnants are regrouping in Pakistan's remote western tribal lands and infiltrating back into Afghanistan to wage a violent insurgency against troops, officials and aid workers.
Pakistan admits to the presence of Taliban members on its territory but denies that they are regrouping or being supported in Pakistan.
The US military said on Saturday that Taliban were trying to destroy President Hamid Karzai's interim government and reinstall their own.