The ability of Legionella pneumophila to cause pneumonia is dependent on intracellular replication within alveolar macrophages. The Icm/Dot secretion apparatus is essential for the ability of L. pneumophila to evade endocytic fusion, to remodel the phagosome by the endoplasmic reticulum (ER), and to replicate intracellularly. Protozoan and macrophage infectivity (pmi) mutants of L. pneumophila, which include 11 dot/icm mutants, exhibit defects in intracellular growth and replication within both protozoa and macrophages. In this study we characterized one of the pmi loci, pmiA. In contrast to the parental strain, the pmiA mutant is defective in cytopathogenicity for protozoa and macrophages. This is a novel mutant that exhibits a partial defect in survival within U937 human macrophage-like cells but exhibits a severe growth defect within Acanthamoeba polyphaga, which results in elimination from this host. The intracellular defects of this mutant are complemented by the wild-type pmiA gene on a plasmid. In contrast to phagosomes harboring the wild-type strain, which exclude endosomal-lysosomal markers, the pmiA mutant-containing phagosomes acquire the late endosomal-lysosomal markers LAMP-1 and LAMP-2. In contrast to the parental strain-containing phagosomes that are remodeled by the ER, there was a decrease in the number of ER-remodeled phagosomes harboring the pmiA mutant. Among several Legionella species examined, the pmiA gene is specific for L. pneumophila. The predicted amino acid sequence of the PmiA protein suggests that it is a transmembrane protein with three membrane-spanning regions. PmiA is similar to several hypothetical proteins produced by bacteria with a type IV secretion apparatus. Importantly, the defect in pmiA abolishes the pore-forming activity, which has been attributed to the Icm/Dot type IV secretion system. However, the mutant is sensitive to NaCl, and this sensitivity is abrogated in the icm/dot mutants. These results suggest that PmiA is a novel virulence factor that is involved in intracellular survival and replication of L. pneumophila in macrophages and protozoan cells.