Several new microorganisms have been isolated from soil samples with high epoxide hydrolase activity toward ethyl 3,4-epoxybutyrate. Screening was performed by enrichment culture on alkenes as sole carbon source, followed by chiral gas chromatography. Eight strains were discovered with enantioselectivity from moderate to high level and identified as bacterial and yeast species. Cells were cultivated under aerobic condition at 30 degrees C using glucose as carbon source and resting cells were used as biocatalysts for kinetic resolution of ethyl 3,4-epoxybutyrate. Among isolated microorganisms, Acinetobacter baumannii showed highest enantioselectivity for (S)-enantiomer, resulting in (R)-ethyl-3,4-epoxybutyrates (> 99%ee, 46% yield). It is the first report on the fact that epoxide hydrolases originating from bacterial species of A. baumannii was applied to kinetic resolution of ethyl 3,4-epoxybutyrate in order to obtain enantiopure high-value-added (R)-ethyl-3,4-epoxybutyrate.