Three thermostable proteases, designated S, N, and B, are extracellular enzymes produced by Bacillus stearothermophilus strain TLS33. They were purified by lysine affinity chromatography, strong anion exchange Q HyperD chromatography, and Ultrogel AcA44 gel filtration. The molecular masses of the enzymes determined by SDS-PAGE and zymography were approximately 36, 53, and 71 kDa, respectively. Thermostable protease S bound strongly to the lysine affinity column and could be purified by this single step. The optimum pH values of proteases S, N, and B were shown to be 8.5, 7.5, and 7.0, respectively. The maximum activities for the enzymes were at 70, 85, and 90 degreesC, respectively. Proteases S, N, and B at pH 7.0 in the presence of 5 mM CaCl2 retained half their activities after 30 min at 72, 78, and 90 degreesC, respectively. All three thermostable proteases were strongly inhibited by the metal chelators EDTA and 1,10-phenanthroline, and the proteolytic activities were restored by addition of ZnCl2. They can thus be classified as Zn2+ metalloproteases. The cleavage specificities of proteases S, N, and B on a 30-residue synthetic peptide from pro-BPN' subtilisin were Tyr-Ile, Phe-Lys, and Gly-Phe, respectively.