Previously, we had isolated by transposon mutagenesis a Legionella pneumophila mutant that appeared defective for intracellular iron acquisition. While sequencing in the proximity of the mini-Tn10 insertion, we found a locus that had a predicted protein product with strong similarity to PilB from Pseudomonas aeruginosa. PilB is a component of the type II secretory pathway, which is required for the assembly of type IV pili. Consequently, the locus was cloned and sequenced. Within this 4-kb region were three genes that appeared to be organized in an operon and encoded homologs of P. aeruginosa PilB, PilC, and PilD, proteins essential for pilus production and type II protein secretion. Northern blot analysis identified a transcript large enough to include all three genes and showed a substantial increase in expression of this operon when L. pneumophila was grown at 30 degrees C as opposed to 37 degrees C. The latter observation was then correlated with an increase in piliation when bacteria were grown at the lower temperature. Southern hybridization analysis indicated that the pilB locus was conserved within L. pneumophila serogroups and other Legionella species. These data represent the first isolation of type II secretory genes from an intracellular parasite and indicate that the legionellae express temperature-regulated type IV pili.