Many endogenous and xenobiotic chemicals are metabolized to epoxides which may be enzymatically hydrated, via microsomal epoxide hydrolase (mEH), to less reactive dihydrodiol derivatives. On the basis of the reported rabbit mEH amino acid sequence [F. S. Heinemann and J. Ozols (1984) J. Biol. Chem. 259, 797-804], we constructed a 35 base oligonucleotide which was used to screen rabbit liver cDNA libraries. Overlapping rabbit mEH clones were isolated and the full-length cDNA sequence of 1653 bp was determined. The rabbit nucleotide sequence has a high degree of similarity (greater than 75%) with cDNA sequences reported for rat and human mEH. Northern blot analyses with fragments of the rabbit cDNA demonstrate that mEH messenger RNA (mRNA) is expressed constitutively in the liver and induced following exposure to phenobarbital or polychlorinated biphenyls. Constitutive expression of mEH mRNA is also observed in rabbit kidney, testes, and lung. Using benzo[alpha]pyrene-4,5-oxide as substrate, mEH enzymatic activity is shown to correlate closely with tissue levels of mEH mRNA. Southern blot analyses of rabbit DNA suggest that the mEH gene exists as a single copy per haploid genome. The mEH amino acid sequences of the human and rat were compared to that of the deduced rabbit protein in order to analyze the degree of conservation and hydropathy profiles in these species. This comparison permitted the formulation of a computer-assisted model of mammalian mEH as it may relate to the microsomal membrane.