Expression of type IV pili (Tfp) correlates with the ability of Neisseria gonorrhoeae to colonize the human host, as well as with adherence to human epithelial tissue, twitching motility, competence for natural transformation, and autoagglutination. N. gonorrhoeae PilF (required for Tfp biogenesis) and PilT (required for twitching motility and transformation) share significant identities with members of a family of putative ATPases involved in membrane trafficking of macromolecules. An open reading frame downstream of the pilT locus encoding a 408-amino-acid protein with 33% identity with the gonococcal PilT protein and 45% identity with the PilU protein in Pseudomonas aeruginosa was characterized, and the corresponding gene was designated pilU. Unlike N. gonorrhoeae pilT mutants, pilU mutants express twitching motility and are competent for DNA transformation. However, loss-of-function mutations in pilU increased bacterial adherence to ME-180 human epithelial cells eightfold and disrupted in vitro Tfp-associated autoagglutination. Comparative alignment of N. gonorrhoeae PilU with other members of the TrbB-like family of traffic ATPases revealed a conserved carboxy-terminal domain unique to family members which are not essential for Tfp biogenesis but which specifically modify Tfp-associated phenotypes. Studies of the pilT-pilU locus by using Northern blotting, transcriptional fusions, and reverse transcription-PCR showed that the two genes encoding closely related proteins with dissimilar effects on Tfp phenotypes are transcribed from a single promoter.