Evidence for the existence in rat and rabbit liver of two microsomal epoxide hydrolases with radically different substrate specificities was obtained, one with a broad specificity (EHb), whilst the other catalyzed the hydrolysis of cholesterol 5 alpha,6 alpha-oxide (EHch), a reaction taken as diagnostic since it was not observed with pure fractions of EHb. The two enzymes were physically separated by immunoprecipitation using antibodies which had been raised against EHb purified to apparent homogeneity. The substrate specificity of the two enzymes is radically different and mutually complementary. Cholesterol 5 alpha,6 alpha-oxide has a trisubstituted oxirane ring. All epoxides of this nature tested to date were not, or very poor, substrates of EHb. The two enzymes can also effectively be discriminated by inhibitors, in that 5 alpha,6 alpha-imino-5 alpha-cholestane-3 beta-ol potently inhibits EHch but not EHb whilst 1,1,1-trichloropropene oxide has the opposite specificity. The cytosolic EH did not significantly contribute to the catalysis of the hydrolysis of cholesterol 5 alpha,6 alpha-oxide.