Limonene-1,2-epoxide hydrolase from Rhodococcus erythropolis DCL14, an enzyme involved in the limonene metabolism of this microorganism, catalyzes the enantioselective hydrolysis of 1-methylcyclohexene oxide. (1R,2S)-1- Methylcyclohexene oxide was the preferred substrate and it was mainly hydrolyzed to (1S,2S)-1-methylcyclohexane-1,2-diol, while (1S,2R)-1- methylcyclohexene oxide was converted more slowly and mainly yielded (1R,2R)- 1-methylcyclohexane-1,2-diol. The reaction proceeded with a high regioselectivity (C1:C2, 85:15). H218O-labelling experiments confirmed that the nucleophile was mainly incorporated at the most substituted carbon atom, suggesting that limonene-1,2-epoxide hydrolase uses an acid-catalyzed enzyme mechanism.