A thermostable D-hydantoinase of thermophilic Bacillus stearothermophilus SD-1 was purified to homogeneity using an immune-affinity chromatography. The affinity chromatography that employed polyclonal antibody immobilized on Sepharose 4B was simple to operate and gave a purification yield of 60% of enzyme activity. Molecular mass of the enzyme was determined to be about 133.9 kDa by gel filtration chromatography and the molecular mass of the subunit was 54 kDa on SDS-PAGE. Mass spectrometric analyses were also performed for the determination of the molecular mass of the native enzyme and its subunit. The apparent molecular masses were 51.1 and 102.1 kDa for the subunit and native enzyme, respectively. Based on the molecular masses determined by these two methods, it is suggested that the D-hydantoinase exists as a dimeric conformation in the cell. Isoelectric pH of the enzyme was observed to be 4.47. It was found that the enzyme requires one manganese ion per molecule of enzyme for the activity. The optimal ph and temperature for the catalytic activity were about 8.0 and 65 degrees C, respectively. The half-life of the enzyme was estimated to be 30 min at 80 degrees C, confirming that the enzyme purified is one of the most thermostable D-hydantoinase reported so far. Kinetic constants of the enzyme for different substrates were also determined.