Abstract A novel, solvent‐free spiking method was used to contaminate foodstuff ( Desmococcus spp. algae) with pyrene for bioassays with the springtail Orchesella cincta L. (Collembola, Entomobryidae). The primary metabolite 1‐hydroxypyrene and two conjugates (pyrene‐1‐glucoside and one tentatively identified as pyrene‐1‐glucosidemalonate) were quantified in the insects by high‐performance liquid chromatography (HPLC) with fluorescence detection. Individuals from an unpolluted site (SUS population) were capable of rapid metabolism of pyrene, reaching an apparent steady state after 24 h with a composite assimilation/transformation rate of approximately 25 pmol pyrene · g −1 fresh weight · h −1 at an exposure level of approximately 18 μg pyrene · g −1 dry weight algae. A cadmium (Cd)‐tolerant population (TOL), genetically distinct from SUS, was exposed in parallel and the populations' pyrene metabolism compared in order to gain insight into any potential cost of Cd‐tolerance. The TOL animals assimilated and/or performed phase 1 transformation of pyrene approximately twice as fast as SUS (∼56 pmol · g −1 · h −1 ), but there was no significant difference in the elimination rate constants between SUS and TOL animals. Attributing this difference to any specific cost of Cd‐tolerance requires more work and may be coincidental to metal tolerance, but pyrene metabolism in these distinct genotypes, subjected to different selection pressures, was nevertheless significantly different.