An NADH-dependent (S)-specific 3-oxobutyryl-CoA reductase from Clostridium tyrobutyricum was purified 15-fold with a yield of 46%. It was homogeneous by gel electrophoresis after three chromatographic steps. The apparent molecular mass was estimated by column chromatography to be 240 kDa. SDS-gel electrophoresis revealed the presence of 33 kDa subunits. Substrates of the enzyme were ethyl and methyl 3-oxobutyrate, 3-oxobutyryl-N-acetylcysteamine thioester, and 3-oxobutyryl coenzyme A. The specific activities were 340 and 10U (mg protein)-1 for the reduction of 3-oxobutyryl coenzyme A and ethyl 3-oxobutyrate, respectively; the Michaelis constants were 300 microM and 300 mM, respectively. The identity of 12 N-terminal amino acid residues was determined. The enzyme was used in a preparative reduction of substrate, yielding ethyl (S)-3-hydroxybutyrate (> 99% enantiomeric excess).