The metabolism of isoprene was investigated with microsomes derived from cell lines expressing human CYP1A1, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2D6, CYP2E1, or CYP3A4. The formation of epoxide metabolites was determined by gas chromatographic analysis. CYP2E1 showed the highest rates of formation of the isoprene monoepoxides 3,4-epoxy-3-methyl-1-butene (EPOX-I) and 3,4-epoxy-2-methyl-1-butene (EPOX-II), followed by CYP2B6. CYP2E1 was the only enzyme showing detectable formation of the diepoxide of isoprene, 2-methyl-1,2:3,4-diepoxybutane. Both isoprene monoepoxides were oxidized by CYP2E1 to the diepoxide at similar enzymatic rates. In order to determine the relative role of CYP2E1 in hepatic metabolism, isoprene as well as the two monoepoxides were also incubated with a series of ten human liver microsomal preparations in the presence of the epoxide hydrolase inhibitor cyclohexene oxide. The obtained activities were correlated with activities towards specific substrates for CYP1A2, CYP2A6, CYP2C9, CYP2D6, CYP2E1 and CYP3A. The results were supportive for those obtained with single human P450 enzymes. Isoprene (monoepoxide) metabolism sowed a significant correlation with CYP2E1 activity, determined as chlorzoxazone 6-hydroxylation. CYP2E1 is therefore the major enzyme involved in hepatic metabolism of isoprene and the isoprene monoepoxides in vitro. To investigae species differences with regard to the role of epoxide hydrolase in the metabolism of isoprene monoepoxides, the epoxidation of isoprene by human liver microsomes was compared to that of mouse and rate liver microsomes. The amounts of monoepoxides formed as a balance between epoxidation and hydrolysis, was measured in incubations with and without the epoxide hydrolase inhibitor cyclohexene oxide. Inhibition of epoxide hydrolase resulted in similar rates of monoepoxide formation in mouse, rat and man. Without inhibitor, however, the total amount of monoepoxides present at the end of the incubation period was twice as high for mouse liver microsomes than for rat and even 15 times as high as for human liver microsomes. Thus, differences in epoxide hydrolase activity between species may be of crucial importance for the toxicity of isoprene in the various species.