D(-)-3-Hydroxybutyrate dehydrogenase (BDH; EC 1.1.1.30) from a poly(D(-)-3-hydroxybutyrate) (PHB) degrading bacterium, Acidovorax sp. SA1, was purified using Toyopearl DEAE-650M, red-Sepharose CL-4B, and Q Sepharose FF. The molecular mass of the enzyme was estimated as 27 kDa by SDS-PAGE and 110 kDa by gel filtration. The gene encoding BDH was cloned and sequenced, and expressed in Escherichia coli. The gene product was purified in two steps with a high yield. The N-terminal amino acid sequence of the enzyme purified from E. coli agreed with that of the purified enzyme from strain SAL The BDH of strain SA1 had high amino acid sequence homology to that of Ralstonia eutropha H16. The K-m values for D(-)-3-hydroxybutyrate and NAD(+) in the oxidation reaction were 4.5x10(-4) M and 8.9x10(5)M, respectively. The K-m values for acetoacetate and NADH in the reduction reaction were 2.4x10(-4)M and 2.9x10(-5)M, respectively.