Two extracellular proteases, E-A and E-B, produced by sporulating cells of Clostridium perfringens NCTC 8798, were isolated by ammonium sulfate fractionation followed by DEAE-Sephacel and Sephacryl S-300 chromatography. E-A was further purified to homogeneity following separation on casein-agarose. E-A and E-B possessed native molecular weights of 330 kDa and 96 kDa respectively. SDS-PAGE of E-A indicated that it was composed of one major 120-kDa subunit. Both E-A and E-B hydrolyzed N-succinyl-L-phenylalanine-p-nitroanilide and were inhibited by chymotrypsin but not antipain or leupeptin, indicating that they were chymotrypsin-like enzymes. Calcium but not dithiothreitol was effective in minimizing inactivation at 50-degrees-C. Comparative analysis of E-A and I-A-1, the principal intracellular protease, indicated that they were very similar but not identical.