Transposon mutagenesis was used to identify genes necessary for the expression of Pseudomonas aeruginosa type 4 fimbriae. In a library of 12 700 mutants, 147 were observed to have lost the spreading colony morphology associated with the presence of functional fimbriae. Of these, 28 had also acquired resistance to the fimbrial-specific bacteriophage P04. The mutations conferring this phage resistance were found to have occurred at at least six different loci, including the three that had been previously shown to be required for fimbrial biosynthesis or function: the structural subunit (pilA) and adjacent genes (pilB,C,D), the twitching motility gene (pilT), and the Sigma 54 RNA polymerase initiation factor gene (rpoN). One novel group of phage-resistant mutants was identified in which the transposon had inserted near sequences that cross-hybridized to an oligonucleotide probe designed against conserved domains in regulators of RpoN-dependent promoters. These mutants had no detectable transcription of pilA and did not produce fimbriae. A probe derived from inverse polymerase chain reaction was used to isolate the corresponding wild-type sequences from a P. aeruginosa PAO cosmid reference library, and two adjacent genes affected by transposon insertions, pilS and pilR, were located and sequenced. These genes were shown to be capable of complementing the corresponding mutants, both at the level of restoring the phenotypes associated with functional fimbriae and by the restoration of pilA transcription. The pilSR operon was physically mapped to Spel fragment 5 (corresponding to about 72–75/0 min on the genetic map), and shown to be located approximately 25 kb from pilA–D. PilS and PilR clearly belong to the family of two-component transcriptional regulatory systems which have been described in many bacterial species. PilS is predicted to be a sensor protein which when stimulated by the appropriate environmental signals activates PilR through kinase activity. PilR then activates transcription of pilA, probably by interacting with RNA polymerase containing RpoN. The identification of pilS and pilR makes possible a more thorough examination of the signal transduction systems controlling expression of virulence factors in P. aeruginosa.