The biotransformation of 2-methylpropene, a gaseous alkene widely used in industry, was investigated in vitro in liver tissue of rats, mice, and humans. Interspecies comparison revealed that the lowest levels of the primary epoxide metabolite were detected in incubations of 2-methylpropene with human liver homogenate, followed by rat and mouse, respectively. Among the human liver samples, however, important interindividual variations were observed. Out of the 16 samples analyzed, only 2 contained measurable epoxide amounts, while in the other samples only traces were detectable. The involvement of rat liver cytochrome P450 2E1 in the activation of 2-methylpropene to its epoxide 2-methyl-1,2-epoxypropane has been established. The lower capacity of the mixed function oxidase system in human liver samples compared to rodents is confirmed. Concerning epoxide detoxifying enzymes, a high microsomal epoxide hydrolase activity was observed in human liver tissue and an intermediate in rat liver, while a low activity was measured in mouse liver. These findings were inversely correlated with the epoxide levels measured in vitro in liver tissue of the three species studied. It can be concluded that, as far as the in vitro metabolism of 2-methylpropene is concerned, neither mouse nor rat represents a good model for the human situation. Although, the same biotransformation pathways are involved, marked quantitative differences in epoxide levels were observed. The results indicate that human liver tissue is exposed in vitro to smaller concentrations of the primary metabolite 2-methyl-1,2-epoxypropane than rodent liver.