An intracellular enzyme, D(-)-fl-hydroxybutyric acid dehydrogenase involved in an intracellular poly-D(-)-fl-hydroxybutyric acid degradation was isolated from a facultative methylotrophic bacterium, P s e u d o m o n a s 135, grown on methanol as a sole carbon and energy source. This enzyme was partially purified to l l.6-fold by ammonium sulphate fractionation and a dye-affinity chromatography. The enzyme catalysed simultaneously the oxidation of D(-)-fl-hydroxybutyric acid (Dfl-HB) and the reduction of acetoacetate. The optimum pH was 8.5 for the oxidation reaction and 5.5-6.0 for the reduction reaction, and the enzyme was stable for 2 weeks at -20 ° C. The Km values for oxidation and reduction reactions were determined as 1.84 mM for Dfl-HB, 0.244 mM for NAD +, 0.319 mM for acetoacetate and 0.032 mM for NADH, respectively. It was also found that D-lactate and NADH significantly inhibited the oxidation reaction by competitive inhibition, and acetoacetate by non-competitive inhibition, respectively. The inhibition constants were determined as 1.49 mM for D-lactate, 0.196mM for NADH and 1.82mM for acetoacetate, respectively. According to an experiment with resting cells, it seemed that the enzyme was constitutive.