Streptococcal pyrogenic exotoxin B (SPE B) was purified and its protease and mitogenic activities were investigated. The apparent molecular mass of SPE B purified in the presence of iodoacetic acid was 42 kDa, whereas 29-kDa SPE B was predominant without the reagent. A polyclonal antibody raised against the 29-kDa species reacted with both species, indicating that the 42-kDa species was a precursor of the 29-kDa entity. Both the 42- and 29-kDa species enhanced [3H]thymidine incorporation into human peripheral blood mononuclear cells, whereas neither had any effect on T cell depleted mononuclear cells. The 29-kDa SPE B possessed caseinolytic activity, with an optimal pH of 8, and the activity was specifically suppressed by the antibody. A group of cysteine protease inhibitors, but no serine-, metallo-, or acidic-protease inhibitors, limited the protease activity, whereas dithiothreitol increased the activity. The DNA sequence around a putative active cysteine residue was identical among the speB genes from Streptococcus pyogenes R70, NY-5, and T19. Taken together, these results indicate that SPE B is identical to a cysteine protease, streptopain (EC 22.214.171.124).