The opportunistic pathogen Pseudomonas aeruginosa produces type 4 fimbriae which promote adhesion to epithelial cells and are associated with a form of surface translocation called twitching motility. Transposon mutagenesis was used to identify loci required for fimbrial assembly or function by screening for mutants that lack the spreading colony morphology characteristic of twitching motility. Six mutants were isolated that contain transposon insertions upstream of the previously characterized gene pilQ. This region contains four genes: pilM-P, which encode proteins with predicted sizes of 37.9, 22.2, 22.8 and 19.0 kDa, respectively. pilM-P appear to form an operon and to be expressed from a promoter in the intergenic region between pilM and the divergently transcribed upstream gene ponA. PilM-P were found to be required for fimbrial biogenesis by complementation studies using twitching motility and sensitivity to fimbrial-specific phage as indicators of the presence of functional fimbriae. This was confirmed by electron microscopy. PilO and PilP did not have homologues in the sequence databases, but the predicted PilN amino acid sequence displayed similarity to XpsL from Xanthamonas campestris, a protein required for protein secretion. PilP contained a hydrophobic leader sequence characteristic of lipoproteins, while PilN and PilO have long internal hydrophobic domains which may serve to localize them to the cytoplasmic membrane. PilM has shared sequence motifs with the cell division protein FtsA from Bacillus subtilis and Escherichia coli, as well as the rod-shape-determining protein MreB from E. coli. These motifs are also conserved in eukaryotic actin, in which they are involved in forming an ATPase domain. Deletion mutants of pilM and pilQ displayed a dominant negative phenotype when transformed into wild-type cells, suggesting that these genes encode proteins involved in multimeric structures.