Proteolytic enzymes inhibit the growth of some strains and opacity variants of Neisseria gonorrhoeae. To understand the inhibitory effects of these enzymes, we examined several strains to determine the actions of proteases on the three predominant proteins in gonococcal outer membranes. namely, the major outer membrane protein (protein I), the sometimes-expressed opaque protein (protein II), and protein III. In a comparison of the protein I species expressed by different strains, we observed a pattern based on subunit molecular weight and susceptibility to enzymatic degradation. Protein I species having molecular weights of 34,000 were more susceptible to proteolysis, whereas protein I species having molecular weights of 33,000 were less susceptible, and protein I species having molecular weights of 32,000 were resistant. This pattern was observed both in intact cells and in purified outer membranes. All of the enzymes degraded protein II, but this susceptibility appeared to be influenced in part by the species of protein I present. Protein III was resistant to all of the proteolytic enzymes tested. Based on the resulting fragments from each proteolytic cleavage of proteins I and II and their membrane associations, we suggest how these proteins may be arranged in intact membranes. Our data suggested the presence of an endogenous gonococcal enzyme. This enzyme appeared to degrade proteins I and II into fragments resembling the fragments resulting from the action of chymotrypsin.