The type 4 pili of Pseudomonas aeruginosa are important cell-associated virulence factors that play a crucial role in mediating (i) bacterial adherence to, and colonization of, mucosal surfaces, (ii) a novel mode of flagella-independent surface translocation known as 'twitching motility', and (iii) the initial stages of the infection process for a number of bacteriophages. A new set of loci involved in pilus biogenesis and twitching motility was identified based on the ability of DNA sequences downstream of the pilG gene to complement the non-piliated (pil) strain, PAO6609. Sequence analysis of a 3.2 kb region directly downstream of pilG revealed the presence of three genes, which have been designated pilH, pilI, and pilJ. The predicted translation product of the pilH gene (13,272 Da), like PilG, exhibits significant amino acid identity with the enteric single-domain response regulator CheY. The putative PilI protein (19,933 Da) is 28% identical to the FrzA protein, a CheW homologue of the gliding bacterium Myxococcus xanthus, and the PilJ protein (72,523 Da) is 26% identical to the enteric methyl-accepting chemotaxis protein (MCP) Tsr. Mutants containing insertions in pilI and pilJ were severely impaired in their ability to produce pili and did not translocate across solid surfaces. The pilH mutant remained capable of pilus production and twitching motility, but displayed an altered motility pattern characterized by the presence of many doughnut-shaped swirls. Each of these pil mutants, however, produced zones that were at least as large as the parent in flagellar-mediated swarm assays. The sequence similarities between the putative pilG, H, I and J gene products and several established chemotaxis proteins, therefore, lend strong support to the hypothesis that these proteins are part of a signal-transduction network that controls P. aeruginosa pilus biosynthesis and twitching motility.