The vacuolar proton pump (V-ATPase) appears to be essential for viability of Dictyostelium cells. To investigate the function of VatM, the 100 kDa transmembrane V-ATPase subunit, we altered its level. By means of homologous recombination, the promoter for the chromosomal vatM gene was replaced with the promoter for the act6 gene, yielding the mutant strain VatMpr. The act6 promoter is much more active in cells growing axenically than on bacteria. Thus, transformants were selected under axenic growth conditions, then shifted to bacteria to determine the consequences of reduced vatM expression. When VatMpr cells were grown on bacteria, the level of the 100 kDa V-ATPase subunit dropped, cell growth slowed, and the A subunit, a component of the peripheral catalytic domain of the V-ATPase, became mislocalized. These defects were complemented by transformation of the mutant cells with a plasmid expressing vatM under the control of its own promoter. Although the principal locus of vacuolar proton pumps in Dictyostelium is membranes of the contractile vacuole system, mutant cells did not manifest osmoregulatory defects. However, bacterially grown VatMpr cells did exhibit substantially reduced rates of phagocytosis and a prolonged endosomal transit time. In addition, mutant cells manifested alterations in the dynamic regulation of cytosolic pH that are characteristic of normal cells grown in acid media, which suggested that the V-ATPase also plays a role in cytosolic pH regulation.