Legionella pneumophila infects humans, causing Legionnaires' disease, from aerosols generated by domestic and environmental water sources. In aquatic environments L. pneumophila is thought to replicate primarily in protozoa. A 'repeats in structural toxin' (RTX) gene, rtxA, from L. pneumophila was identified recently that plays a role in entry and replication in human macrophages and also has the ability to infect mice. However, the role of this gene in the interaction of L. pneumophila with environmental protozoa and its distribution in different Legionella species has not been examined. Southern analyses demonstrated that rtxA is present in all L. pneumophila isolates tested and correlates with species that have been shown to cause disease in humans. To evaluate the importance of rtxA in the interaction with protozoa a series of studies was carried out in an environmental host for L. pneumophila, Acanthamoeba castellanii. The L. pneumophila rtxA gene plays a role in both adherence and entry into A. castellanii similar to that observed in human monocytic cells. Furthermore, it was found that rtxA is involved in intracellular survival and trafficking. In addition to demonstrating involvement of rtxA in the interaction of L. pneumophila with host cells, these data support a role for this gene both during disease in humans and in environmental reservoirs.