3,4-Dihydrocoumarin hydrolase (DCH) from Acinetobacter calcoaceticus F46, which was previously found on screening for aromatic lactone-hydrolyzing enzymes, catalyzes the hydrolysis of several linear esters. The substrate specificity of the enzyme toward linear esters was quite characteristic, i.e., (1) it was specific toward methyl esters, (2) it recognized the configuration at the 2-position, and (3) it hydrolyzed diesters to monoesters. DCH hydrolyzed the methyl esters of β-acetylthioisobutyrate and cetraxate. The products of these reactions were identified as D-β-acetylthioisobutyrate and cetraxate, respectively, i.e., the hydrolysis reactions catalyzed by DCH were stereo- and/or regioselective. With recombinant Escherichia coli cells expressing the DCH gene as a catalyst, stereospecific hydrolysis of methyl β-acetylthioisobutyrate and regioselective hydrolysis of methyl cetraxate proceeded efficiently.