The transcriptional activator PhoP is important for survival of Yersinia pestis in macrophage phagosomes. However, the phagosomes inhabited by Y. pestis have not been well characterized, and the mechanism by which PhoP promotes bacterial survival in these vacuoles is not fully understood. Lysosomal tracers, as well as antibodies to late endosomal or lysosomal proteins, were used in conjunction with confocal or electron microscopy to study the trafficking of phagosomes containing phoP(+) or phoP mutant Y. pestis strains or latex beads in J774A.1 macrophages. Phagosomes containing phoP(+) or phoP mutant Y. pestis acquired lysosomal markers to the same degree that phagosomes containing latex beads acquired these markers after 1.5 h of infection, showing that nascent phagosomes containing Y. pestis fuse with lysosomes irrespective of the phoP genotype. Similar results were obtained when phagosomes containing viable or dead phoP(+) Y. pestis cells or beads were analyzed at 8 h postinfection, indicating that the Y. pestis vacuole does not become secluded from the lysosomal compartment. However, only viable phoP(+) bacteria induced the formation of spacious phagosomes at 8 h postinfection, suggesting that Y. pestis can actively direct the expansion of its vacuole. PhoP-regulated genes that are important for survival of Y. pestis in phagosomes were identified by Tn5-lacZ mutagenesis and oligonucleotide microarray analysis. Three such genes were identified, and the products of these genes are predicted to promote resistance to antimicrobial peptides (ugd and pmrK) or low-Mg(2+) conditions (mgtC) found in phagosomes. Viable count assays carried out with Y. pestis ugd, mgtC, and ugd mgtC mutants revealed that the products of ugd and mgtC function independently to promote early survival of Y. pestis in macrophage phagosomes.