To investigate the involvement of RelA in the regulation of Legionella pneumophila virulence, a deletion substitution was constructed in the relA gene. The relA knockout resulted in an undetectable level of ppGpp in the cells during the stationary phase, but the original level was restored when the relA gene product was supplied on a plasmid. The effect of the relA mutation was examined with two systems that are known to be expressed during the stationary phase in L. pneumophila. Pigment production was found to be dependent on the relA gene product, and only one-half as much pigment was produced by the relA mutant as by the wild-type strain. Flagellum gene expression was also found to be dependent on the relA gene product, as determined with a flaA::lacZ fusion. However, the relA gene product was found to be dispensable for intracellular growth both in HL-60-derived human macrophages and in the protozoan host Acanthamoeba castellanii. To determine the involvement of the relA gene product in expression of L. pneumophila genes required for intracellular growth (icm/dot genes), nine icm::lacZ fusions were constructed, and expression of these fusions in the wild-type strain was compared with their expression in relA mutant strains. Expression of only one of the icm::lacZ fusions was moderately reduced in the relA mutant strain. Expression of the nine icm::lacZ fusions was also examined in a strain containing an insertion in the gene that codes for the stationary-phase sigma factor RpoS, and similar results were obtained. We concluded that RelA is dispensable for intracellular growth of L. pneumophila in the two hosts examined and that both RelA and RpoS play minor roles in L. pneumophila icm/dot gene expression.