Legionella pneumophila expresses pili of variable lengths, either long (0.8 to 1.5 microm) or short (0.1 to 0.6 microm), that can be observed by transmission electron microscopy. We have identified a gene in L. pneumophila with homology to the type IV pilin genes (pilEL). An insertion mutation was constructed in pilEL and introduced into the L. pneumophila wild-type strain by allelic exchange. The pilin mutant is defective for expression of long pili. Reintroduction of the pilin locus on a cosmid vector restores expression of the long pili. The L. pneumophila pilEL mutant exhibited approximately a 50% decrease in adherence to human epithelial cells (HeLa and WI-26 cells), macrophages (U937 cells), and Acanthamoeba polyphaga but had a wild-type phenotype for intracellular replication within these cells. Southern hybridization analysis showed that the pilEL locus is present in L. pneumophila serogroups 1 through 13 but is variable in 16 other Legionella species. The presence of a type IV pilin gene and its expression by L. pneumophila may provide an advantage for colonization of lung tissues during Legionnaires' disease and invasion of amoebas in the environment.