Studies of the interaction of Legionella pneumophila with free-living amoebae showed that Naegleria lovaniensis and Acanthamoeba royreba could use L. pneumophila as a sole food source. However, growth of the amoebae on nonnutrient agar plates seeded with L. pneumophila was slower than growth on nonnutrient agar plates seeded with Escherichia coli. On inoculation of L. pneumophila into axenic cultures of N. lovaniensis and A. royreba, 99.9% of the L. pneumophila was destroyed within 24 h. After several weeks, however, some amoeba cultures became chronically infected and supported the growth of L. pneumophila. Amoebae exposed to L. pneumophila and containing adhered L. pneumophila, L. pneumophila antigens, or both, showed no increased pathogenic potential on intranasal inoculation of weanling mice. Similarly, L. pneumophila propagated in chronically infected amoeba cultures showed no increase in virulence on intraperitoneal inoculation of guinea pigs relative to L. pneumophila grown in yeast extract broth.