A study was conducted to determine the cytochrome (CYP) P450 enzymes responsible for the bioactivation of aflatoxin B1 (AFB1) into its epoxide form (AFBO) in duck liver microsomes. Six male and six female 6-week-old Pekin ducks were used. The biochemical toxicology strategies applied included the use of selective inhibitors, prototype substrate activity for specific human P450s, correlation between aflatoxin bioactivation and enzymatic activity of prototype substrates, and the expression of specific CYP450 enzymes using antibodies against human CYP450s. Enzymatic activity was detected for the duck orthologues CYP1A1/2, CYP2A6 and CYP3A4 but not for the CYP2D6 orthologue. Immunoreactive proteins for CYP1A1, CYP2A6 and CYP3A4 were also detected. Inhibition studies suggested that the duck turkey CYP2A6 orthologue and, to a lesser extent, the CYP1A1 orthologue are involved in the bioactivation of AFB1. Correlation studies, however, suggest that CYP3A4, CYP2A6 and CYP1A1/2 are all involved in AFBO formation. The finding that four CYP enzymes may be involved in AFB1 bioactivation in ducks could explain the high sensitivity of this species to AFB1. Further studies are needed to fully elucidate the phase I hepatic metabolism of AFB1 in ducks, the only poultry species that develops hepatic cancer from AFB1 exposure.