1-Chloro-2-hydroxy-3-butene (CHB) is an in vitro metabolite of 1,3-butadiene, a rodent/human carcinogen. To search for an approach detecting CHB in vivo, it is vital to obtain a full understanding of CHB metabolism. Previously, we demonstrated that CHB was bioactivated to 1-chloro-3-buten-2-one (CBO) by alcohol dehydrogenase. However, CHB metabolism by cytochrome P450s has not been reported. Thus, in the present study, CHB metabolism by rat liver microsomes was investigated. The results showed that CHB was converted to 1-chloro-3,4-epoxy-2-butanol (CEB) and CBO. 4-Methylpyrazole, a cytochrome P450 2E1-specific inhibitor, inhibited the formation of both CEB and CBO, while 1-benzylimidazole, a generic cytochrome P450 inhibitor, completely abolished the formation of CEB and CBO, suggesting that CHB metabolism was mediated by cytochrome P450s. Because the molecules have two chiral centers, CEB was detected as two stereoisomers, which were designated D-CEB and M-CEB, and were characterized as (2S,3R)-/(2R,3S)-CEB and (2R,3R)-/(2S,3S)-CEB, respectively. The amounts of M-CEB were more than those of D-CEB by 50-80%. The amounts of CEB and CBO increased linearly over time from 10 (or 20 min for CBO) to 50 min. CHB metabolism followed Michaelis-Menten kinetics; the Km and Vmax values were determined to be 6.4 ± 0.7 mM and 0.10 ± 0.01 nmol/min/mg protein for D-CEB, 4.2 ± 0.5 mM and 0.16 ± 0.01 nmol/min/mg protein for M-CEB, and 4.0 ± 0.5 mM and 4.6 ± 0.5 nmol/min/mg protein for CBO, respectively. Thus, CBO was the dominant product of CHB metabolism. Moreover, CEB was genotoxic at ≥ 50 μM as evaluated by the comet assay. Collectively, the data showed that CHB could be bioactivated to CEB and CBO by cytochrome P450s with CBO being the predominant product. Thus, the formation of CEB and CBO can be used as evidence of CHB production. The products may also play a role in toxicity of CHB.