The phase-variable PilC proteins of pathogenic Neisseria species have recently been implicated in both assembly and cellular adherence functions of the type 4 pili of these pathogens. We describe here the cloning of full-length pilC1 and pilC2 genes and the complete sequencing of the pilC2 gene of Neisseria gonorrhoeae MS11. Sequential inactivation of both genes by gene replacement in piliated (P+) variants of N. gonorrhoeae MS11 led initially to a non-piliated (P-) phenotype; however, spontaneous P+ variants could be derived from some pilC1,2 double mutants which produced morphologically intact pili. Purified pili from pilC1,2 mutants revealed no detectable PilC protein. Instead, a novel protein about 70 kDa in size appeared in the pili preparations of P+ mutants; this protein exhibited no immunological cross-reactivity with PilC1 or PilC2. We propose that this novel factor replaces the function of PilC in pilus biogenesis. Using isogenic N. gonorrhoeae strains which produce identical PilE (pilin) proteins we demonstrate that pili associated with the 70 kDa protein do not confer gonococcal adherence to human epithelial cells, in contrast to pili assembled in the presence of PilC1 or PilC2.