A thermophilic bacterium that can grow on poly(3-hydroxybutyrate) (PHB) was isolated from a hot spring. The Isolate, designated strain HS, was a gram-negative aerobic rod and grew optimally at 45-50 degrees C. From its principal phenotypic characteristics, strain HS was revealed to be closely related to Leptothrix discophora. Sequence analysis of the 16S rRNA gene also supported this result. PHB depolymerase was purified to electrophoretic homogeneity from cell-free culture broth by hydrophobic interaction chromatography using Toyopearl HW-55F in combination with ammonium sulfate precipitation. The molecular weight of the enzyme was calculated to be 4.6 x 10(4) by electrophoresis and 4.3 x 10(4) by gel filtration. The enzyme was stable up to 65 degrees C and the optimum reaction temperature was 70 degrees C. A K-m of 0.018 g/l was determined for PHB at 70 degrees C. An isoelectric point of pH 8.6 was obtained by isoelectric focusing. The enzyme activity was inhibited by dithiothreitol but not by phenylmethylsulfonyl fluoride. The N-terminal amino acid sequence was unlike that of any other bacterial PHB depolymerase. Secretion of the enzyme was accelerated by the addition to the culture medium of 3-hydroxybutyrate which is the product of the enzyme.