Microbial asymmetric oxidation of 2-butyl-1,3-propanediol was investigated for an efficient synthesis of S- and R-enantiomers of 2-hydroxymethylhexanoic acid (2-HMHA). From an intensive survey of the stocked bacterial strains, Acetobacter pasteurianus IAM 12073 and Pseudomonas putida IFO 3738 were found to show the highest S- and R-2-HMHA-producing activity, respectively. Under optimized conditions, A. pasteurianus (351 mg dry cell weight) and P putida (642 mg dry cell weight) cells produced 12.0 g l(-1) S-2-HMHA with 89% enantiomeric excess (e.e.) at 24 h of incubation and 5.1 g l(-1) R-2-HMHA with 94% e.e. at 35 h of incubation from 2-butyl-1,3-propanediol.