Histoplasma capsulatum (Hc) is a facultative intracellular fungus that modulates the intraphagosomal environment to survive within macrophages (Mφ). In the present study, we sought to quantify the intraphagosomal pH under conditions in which Hc yeasts replicated or were killed. Human Mφ that had ingested both viable and heat-killed or fixed yeasts maintained an intraphagosomal pH of∼ 6.4–6.5 over a period of several hours. These results were obtained using a fluorescent ratio technique and by electron microscopy using the 3-(2, 4-dinitroanilo)-3′-amino-N-methyldipropylamine reagent. Mφ that had ingested Saccharomyces cerevisae, a nonpathogenic yeast that is rapidly killed and degraded by Mφ, also maintained an intraphagosomal pH of∼ 6.5 over a period of several hours. Stimulation of human Mφ fungicidal activity by coculture with chloroquine or by adherence to type 1 collagen matrices was not reversed by bafilomycin, an inhibitor of the vacuolar ATPase. Human Mφ cultured in the presence of bafilomycin also completely degraded heat-killed Hc yeasts, whereas mouse peritoneal Mφ digestion of yeasts was completely reversed in the presence of bafilomycin. However, bafilomycin did not inhibit mouse Mφ fungistatic activity induced by IFN-γ. Thus, human Mφ do not require phagosomal acidification to kill and degrade Hc yeasts, whereas mouse Mφ do require acidification for fungicidal but not fungistatic activity.