1,3-Butadiene (BD) is a gas used widely in the rubber and plastics industry as an intermediate in production processes and has been detected in automobile exhaust and cigarette smoke. BD requires metabolic activation to exert toxicity and has been shown to be carcinogenic in rodents. IARC has classified BD as a group 2A (probably carcinogenic to humans) carcinogen. The initial oxidation of BD to butadiene monoxide (BMO) occurs primarily via cytochrome P450 2E1 and two stereoisomers of BMO (R and S) can be formed. (R) and (S)-BMO are metabolized differently and demonstrate markedly different toxicities in isolated rat hepatocytes. This work examined the generation of (R) and (S)-BMO from BD by cytochrome P450 2E1 from rabbit, rat and human. BMO level was measured by GC-MS analysis and enantiomeric composition was determined by GC-FID. The greatest rate of formation of BMO from BD was obtained with rabbit cytochrome P4502E1 followed by human and then by rat. Enantiomeric distribution of R and S-BMO produced by the three species demonstrated no significant differences.