Previously, we provided evidence for the involvement of multiple cytochrome P450 enzymes in the metabolism of 1,3-butadiene, a rodent and possibly a human carcinogen, to butadiene monoxide in mouse and rat liver microsomes. In this study, 1,3-butadiene oxidation by seven cDNA-expressed human P450 enzymes and by human, mouse, and rat liver microsomes was characterized. Incubations with cDNA-expressed human P450 1A2, 2A6, 2B6, 2D6, and 2E1 resulted in the formation of butadiene monoxide, whereas incubations with P450 1A1 and 3A4 did not lead to the detection of any metabolite. Of the active isozymes examined, P450 2A6 and 2E1 were the most active when butadiene monoxide formation rates were normalized for the P450 content of the microsomes. With six human liver microsomal samples, 1,3-butadiene oxidation exhibited nearly threefold individual variation in the amounts of butadiene monoxide detected, and butadiene monoxide formation was NADPH- and time-dependent and was inhibited by the addition of 1-benzyl-imidazole or 4-methylpyrazole, known cytochrome P450 inhibitors. Correlation studies provided evidence for major roles by P450 2A6 and 2E1 in 1,3-butadiene oxidation in human liver microsomes. Butadiene monoxide formation rates in human liver microsomes were similar, or higher, than the rate obtained in mouse liver microsomes, whereas 1,3-butadiene oxidation rates in human and mouse liver microsomes were higher than the rate obtained in rat liver microsomes. These results provide direct evidence that 1,3-butadiene is a substrate for multiple P450 enzymes and suggest that humans may be at higher risk of expressing 1,3-butadiene toxicity compared to mice or rats. In addition, these results suggest that the mouse may be the more appropriate animal model to assess human risk.