A previously undiscovered intracellular serine protease activity, which we have called intracellular serine protease-4, was identified in extracts of stationary Bacillus subtilis cells, purified 260 fold from the cytoplasmic fraction, and characterized. The new protease was stable and active in the absence of Ca2+ ions and hydrolyzed azocasein and the chromogenic substrate carbobenzoxy-carbonyl-alanyl-alanyl-leucyl-p-nitroanilide, but not azocollagen or a variety of other chromogenic substrates. The protease was strongly inhibited by phenylmethylsulfonylfluoride, chymostatin and antipain, but not by chelators, sulfhydryl-reactive agents or trypsin inhibitors. Its activity was stimulated by Ca2+ ions and gramicidin S; its pH and temperature optima were 9.0 and 37 degrees C, respectively. Although intracellular serine protease-4 was immunochemically distinct from intracellular serine protease-1, it was absent from a mutant in which the gene encoding the latter was disrupted.