Monitoring parameters for the assessment of oil and gas related contaminants and their biological effects need validation before application in the Arctic. For such monitoring purposes, we evaluated the potential use of three biomarkers (acetylcholinesterase, acyl-CoA oxidase and glutathione S-transferase) for application to an Arctic bivalve (Astarte borealis) and determined the body residue of pyrene and two pyrene metabolites (1-hydroxypyrene and pyrene-1-glucuronide) in Arctic benthic species (bivalve: Macoma calcarea; polychaete: Nephtys ciliata) and temperate benthic species (bivalve: Limecola balthica; polychaete: Alitta virens) in order to establish the potential of polycyclic aromatic hydrocarbons (PAHs) metabolite profiles as biomarkers of exposure in such species. Experimental PAH exposure levels were probably too low (0.2–1.7 mg/kg dry weight in sediment) to induce or inhibit biomarker responses in A. borealis. Concentrations of pyrene and pyrene metabolites varied between species, although no consistent patterns could be established among taxonomic groups and locations. Metabolites made up to 79% of the total pyrene concentrations, indicating that basal metabolic activity is affecting pyrene kinetics even at low concentrations in all species. This indicates that Arctic and temperate species could show similar metabolism patterns of PAHs, although more insight into the effects of confounding factors is needed.