We examined the role of the flagella of Legionella pneumophila in the infection of amoebae and human monocyte-like cells. Insertional mutants were constructed with mini-Tn10. Ten mutants (F-) which did not react with polyclonal L. pneumophila antiflagellar antisera were identified. Ten randomly selected mutants (F+) that did react with the polyclonal antiflagellar antiserum were also identified. The infectivity of these 20 mutants in Hartmannella vermiformis and human U937 cells was characterized. Seven of the 10 F- mutants were attenuated in their ability to multiply in the amoebae during the first 3 days of coincubation and failed to multiply in U937 cells. Three of the 10 F- mutants multiplied as well as the wild-type parent strain did in amoebae and to a limited degree in U937 cells. None of the 10 F+ mutants were attenuated in either the amoebae or U937 cells. While the flagellar structure is not essential for virulence, the ability of L. pneumophila to infect amoebae and human phagocytic cells appears to be linked to flagellar expression. We believe that the attenuated F- mutants contain insertions in genes critical to both flagellum expression and the infection process.