A thermostable D-hydantoinase was isolated from thermophilic Bacillus thermocatenulatus GH-2 and purified to homogeneity by using immunoaffinity chromatography. The molecular mass of the enzyme was determined to be about 230 kDa, and a value of 56 kDa was obtained as a molecular mass of the subunit on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, implying that oligomeric structure of the enzyme is tetrameric. Isoelectric pH of the enzyme was found to be approx 4.3. The enzyme required Mn2+ for the activity and exhibited its highest activity with phenylhydantoin as a substrate. The optimal pH and temperature for catalytic activity were about 7.5 and 65 degrees C, respectively. The half-life of the enzyme was estimated to be about 45 min at 80 degrees C.