D(-)-beta-Hydroxybutyrate dehydrogenase was purified from Zoogloea ramigera I-16-M to electrophoretic homogeneity. The molecular weight of the enzyme as determined by Sephadex G-200 gel filtration was 112,000, and the monomer molecular weight estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate was 28,000, indicating that the native enzyme is a tetramer with four identical subunits. The enzyme showed a pH optimum at 8.0 in the oxidation reaction, and a broad pH optimum (5.5-7.5) in the reduction reaction. The Km values for D(-)-beta-hydroxybutyrate and NAD in the oxidation reaction were 3.2 X 10(-4) M and 5.7 X 10(-5) M, respectively. The Km value for acetoacetate in the reduction reaction was 1.5 X 10(-4) M and that for NADH was 1.5 X 10(-5) M. Acetyl CoA, D-lactate, and 2-hydroxybutyrate were effective inhibitors for the oxidation of D(-)-beta-hydroxybutyrate. The enzyme was sensitive to the inhibitory actions of sulfhydryl reagents such as p-chloromercuribenzoic acid, 5,5'-dithiobis(2-nitrobenzoic acid) and HgCl2.