Ambient inclusion trails (AIT) are enigmatic microtubular structures created by the migration of mineral crystals through a lithified substrate. The decomposition of organic material has been suggested as the driving force for the crystal migration, but has yet to be rigorously tested. AIT may hold potential as a biosignature for investigating early life on Earth if the associated organic material can be shown to be biological. This paper attempts to test the formation mechanism and biogenicity of AIT from the c. 3400 Ma Strelley Pool sandstone of Western Australia using NanoSIMS technology. In doing so, we demonstrate the unique ability of the NanoSIMS to combine sub-micron scale imaging with in situ chemical and isotopic data, thereby enhancing our ability to evaluate the biogenicity criteria for Archaean microstructures. Enrichments of a suite of major elements (C, N, P, S) and trace elements (Co, Fe, Ni, Zn), often associated with biological processes, are found within several AIT in this sandstone. C and N enrichments are most common along AIT margins, and correlate with depletions of Si, O, Ca and Mg, indicating that this material is indeed organic in nature. Î´^sup 13^C values of this carbonaceous material average -26[per thousand]. Petrographic observations show that some of the AIT occur in the centre of detrital sandstone grains where they were sealed from later fluid flow and therefore preserve primary Archaean (bio)geochemistry. In contrast, AIT found around the outer edges of sandstone grains may contain more recent organic material introduced by later fluid migration and are an unreliable biosignature. Using the petrographic and geochemical data a multi-stage model for AIT formation and subsequent diagenetic modification is proposed. The possible sources of the primary organic material are discussed and we conclude that the data are consistent with a biological origin for these AIT. An abiogenic origin is more difficult to sustain but cannot yet be completely excluded for AIT in general.