Salmonella species are facultative intracellular pathogens. Following entry into mammalian host cells, they reside in membrane-bound vacuoles, resist killing, and replicate. In this work, we investigated the importance of phagosomal pH in the ability of Salmonella typhimurium to survive and replicate within macrophages. Intraphagosomal pH was measured in situ by recording the fluorescence intensity of a pH-sensitive probe, DM-NERF dextran. The majority of vacuoles containing S. typhimurium (live, heat killed, or formalin fixed) acidified from pH > or = 6.0 to between pH 4.0 and 5.0 within 60 min after formation. In contrast, Mycobacterium avium-containing vacuoles failed to acidify even at later time points. Acidification of S. typhimurium-containing vacuoles was completely blocked by treatment of host cells with bafilomycin A, a specific inhibitor of vacuolar proton-ATPases. Bafilomycin inhibition of vacuolar acidification from the onset of infection significantly decreased the survival of S. typhimurium in macrophages. Furthermore, bafilomycin treatment at 2, 4, 8, or even 12 h postinfection decreased the percentage of recoverable bacteria by up to 20-fold. Loss of bacterial viability was seen with several other reagents which, like bafilomycin, raise the pH of phagosomal compartments but are not directly lethal to the bacteria or host cells. Thus, we conclude that Salmonella-containing phagosomes acidify soon after formation and hypothesize that an acidic environment is necessary for survival and replication of the bacteria within the macrophage.