Type 4 fimbriae of Pseudomonas aeruginosa are surface filaments involved in host colonization. They mediate both attachment to host epithelial cells and flagelia-independent twitching motility. Four additional genes, pilW, pilX, pilY1 and pilY2, are located on Spel fragment E in the 5 kb intergenic region between the previously characterized genes pilV and pilE, which encode prepilin-like proteins involved in type 4 fimbrial biogenesis. The phenotypes of a transposon insertion and other mutations constructed by allelic exchange show that these genes are involved in the assembly of type 4 fimbriae. The PilW and PilX proteins are membrane located, possess the hydrophobic N-terminus characteristic of prepilin-like proteins, and appear to belong to the GspJ and GspK group of proteins that are required for protein secretion in a wide range of Gram-negative bacteria. These findings increase the similarities between the fimbrial biogenesis and the Gsp-based protein-secretion supersystems. PilY1 is a large protein with C-terminal homology to the PilC2 protein of Neisseria gonorrhoeae, thought to be a fimbrial tip-associated adhesin, and which, like PilY1, is involved in fimbrial assembly. PilY1 appears to be located in both the membrane and the external fimbrial fractions. PilY2 is a small protein that appears to play a subtle role in fimbrial biogenesis and represents a new class of protein.