Iran says it independently collected samples at a suspect military site where illicit nuclear work is alleged to have occurred and later handed them to the UN's absent inspectors.
The disclosure on Monday that international monitors were not physically present is likely to feed critics of a nuclear deal between Iran and world powers, who have poured scorn on measures used to check if Tehran's atomic program is peaceful.
In a mark of the high stakes at play it drew a quick reaction from the UN's International Atomic Energy Agency, whose chief insisted that "the integrity of the sampling process and the authenticity of the samples" was not compromised.
The samples were taken under "established procedures", IAEA director general Yukiya Amano said, noting "significant progress" is being made in its long-running probe of whether Iran ever sought to develop a nuclear bomb.
The site at Parchin, east of Tehran, has been at the centre of international scepticism of Iran's activities, specifically that as late as 2003 it carried out work there aimed at developing an atomic weapon.
Iran says accusations from Western intelligence agencies - including that it conducted explosives tests at Parchin - are groundless and based on malicious information provided by its enemies.
The sample taking is linked to a so-called roadmap with a December 15 deadline by when the IAEA says ambiguities about past possible military dimensions of Iran's nuclear activities must be resolved.
Much work remains to be done on that report, Amano said.
Iran's environmental sampling from specific parts of the Parchin complex took place in the past week.