Three gonococcal genes have been identified which encode proteins with substantial similarities to known components of the type IV pilus biogenesis pathway in Pseudomonas aeruginosa. Two of the genes were identified based on their hybridization with a DNA probe derived from the pilB gene of P. aeruginosa under conditions of reduced stringency. The product of the gonococcal pilF gene is most closely related to the pilus assembly protein PilB of P. aeruginosa while the product of the gonococcal pilT gene is most similar to the PilT protein of P. aeruginosa which is involved in pilus-associated twitching motility and colony morphology. The products of both of these genes display canonical nucleoside triphosphate binding sites and are predicted to be to cytoplasmically localized based on their overall hydrophilicity. The gonococcal pilD gene, identified by virtue of its linkage to the pilF gene, is homologous to a family of prepilin leader peptidase genes. When expressed in Escherichia coli, the gonococcal PilD protein functions to process gonococcal prepilin in a manner consistent with its being gonococcal prepilin peptidase. These results suggest that Neisseria gonorrhoeae is capable of expressing many of the essential elements of a highly conserved protein translocation system and that these gene products are probably involved in pilus biogenesis.