In this study, temporal changes in the extractability of 14C-pyrene, at native concentrations, were followed in two soils with differing organic matter contents, under sterile and non-sterile conditions over 24 weeks by a sequential solvent extraction scheme. No significant loss of the added 14C-pyrene was observed during the incubation. Significant decreases in methanol:water and n-butanol extractability were observed with increasing soil–pyrene contact time. Significant non-extractable residues were formed in all soils, with the largest increases found in the non-sterile soils. After 8 weeks soil–pyrene contact time, there was a significant increase in the rate and extent of sequestration of pyrene in the biologically active soils. This indicated that the aging of pyrene was initially a physical process, with active microbial communities increasing the rate and extent of residue formation after 8 weeks soil–pyrene contact time. These findings suggest that there is a need for longer term ageing experiments following the role of microbial communities on the formation of solvent non-extractable residues. The humin fraction of the soil organic matter contained the majority of the 14C-pyrene associated activity which was not extractable using the scheme of sequential solvents. Saponification of the soil humin resulted in the release of similar amounts of 14C-pyrene associated activity from sterile and non-sterile soils. Solvent extraction with methanol:water was found to significantly underestimate the bioavailable fraction, whereas n-butanol overestimated the bioavailability of the 14C-pyrene-associated activity when assessed by bacterial mineralization after 24 weeks soil–pyrene contact time.