Previously we have reported that Legionella pneumophila can mediate plasmid DNA transfer at a frequency of about 10−3 transconjugants per donor and that this process is dependent on several icm genes. Here we characterize the icm-dependent conjugal ability of L. pneumophila and study its relationship to intracellular multiplication and host cell killing. We found that three icm genes and the RSF1010 mobA gene are completely required and that three icm genes and the RSF1010 mobC gene are partially required for conjugation. Conjugation occurred during lag phase and stopped when the cell number increased. Inhibition of transcription or translation in the donor had only a minor effect on conjugation frequency. These results suggest that stationary-phase bacteria contain a functional icm complex that can mediate conjugal DNA transfer and probably can initiate infection of human macrophages as well. We also found that a functional RSF1010 mobilization system inhibits intracellular multiplication and killing of human macrophages by L. pneumophila. The strongest inhibition was observed in icm insertion mutants complemented with wild-type icm genes on an RSF1010-derived plasmid. These results suggest that the conjugation substrate probably competes with the natural substrate of the L. pneumophila icm system for transfer outside the bacterial cell. We propose that the function of the L. pneumophila icm system is to transfer effector molecules to the host cell. These effector molecules may interact with components of the host cell that are involved in phagosome formation and fate.