Abstract Sediment contaminated with polycyclic aromatic hydrocarbons (PAHs) from a Superfund site on the Elizabeth River (ER) in Portsmouth, VA, is teratogenic to embryonic killifish ( Fundulus heteroclitus) from reference sites. However, embryos born to a population of ER killifish are resistant to PAH-induced teratogenicity. Mechanisms underlying the resistance are unclear; however, ER killifish are refractory to induction of metabolic enzyme cytochrome P4501A (CYP1A), at the level of mRNA, protein and activity. The contaminated ER sediment comprises a complex mixture of PAHs with different mechanisms of toxicity. While many are inducers of metabolic enzymes involved in both phase I and phase II of biotransformation, some PAHs can also inhibit phase I enzymatic activity. Previous research has shown that co-exposure to PAHs with different modes of action can result in synergistic embryotoxicity (Billiard, S.M., Meyer, J.N.D., Wassenberg, M., Hodson, P.V., Di Giulio, R.T., 2008. Nonadditive effects of PAHs on early vertebrate development: mechanisms and implications for risk assessment. Toxicological Sciences 105, 5–23). Two of the abundant PAHs at the ER are fluoranthene (FL), a CYP1A inhibitor, and benzo[a]pyrene (BaP), a CYP1A inducer. Based on the ER resistant phenotype and the PAH mixture in the ER sediment, we hypothesized that the inhibition of CYP1A activity affects the teratogenicity of PAHs through a biotransformation-mediated mechanism. To examine this hypothesis, we compared the responses of killifish embryos born to parents from the ER and from a reference site (King's Creek (KC), VA) after a water-borne exposure to BaP (0–400 μg/L) in the presence or absence of FL (0–500 μg/L). Embryos were dosed from 24 to 120 h post-fertilization (hpf) and were analyzed for induction of CYP1 enzymatic activity as measured by the ethoxyresorufin-O-deethylase (EROD) assay, cardiac deformities, and BaP metabolic profile. KC embryos showed significant induction of CYP1 protein activity at all BaP concentrations examined. Co-exposure to 500 μg/L of FL significantly decreased CYP1 activity and increased cardiac deformities. ER embryos showed no change in CYP1 activity or cardiac deformities for any treatment. Significantly greater concentrations of BaP and BaP 9,10-dihydrodiol were recovered from ER embryos compared to those from KC. Co-exposure with FL did not significantly alter the amount of BaP or the metabolites recovered in either population. These findings suggest that the teratogenicity observed by co-exposure to BaP and FL cannot fully be explained by alteration in BaP metabolism. This study also indicates that the metabolic adaptation observed in the ER killifish cannot be explained simply by the refractory CYP1 phenotype.