Of the total ATPase activity in homogenates of the ameba, Dictyostelium discoideum, approximately one-third was inhibited at pH 7 by 25 microM 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). Upon isopycnic sucrose density gradient centrifugation, the bulk of the NBD-CI-sensitive ATPase activity was recovered in a major membrane fraction with a broad peak at 1.16 g/ml, well-resolved from markers for plasma membranes, mitochondria, lysosomes and contractile vacuoles. The gradient peak had a specific activity of 0.5 mumol/min per mg protein. The activity was half-inhibited by 1 microM silicotungstate, 2 microM diisothiocyanatostilbene disulfonate (DIDS), 2.5 microM dicyclohexylcarbodiimide (DCCD), 4 microM NBD-CI and 20 microM N-ethylmaleimide (NEM) but was resistant to conventional inhibitors of mitochondrial and plasma membrane ATPase. That this ATPase activity constituted a proton pump was shown by the MgATP-dependent uptake and quenching of Acridine orange fluorescence by partially purified vacuoles. The Acridine orange uptake was specifically blocked by the aforementioned inhibitors. The generation of proton electrochemical gradients was suggested by the stimulation of enzyme activity by protonophores (fatty acids) and cation exchangers (nigericin). Uncoupling stimulated the ATPase activity as much as 20-fold, revealing an unusually high impermeability of the membranes to protons. ATPase activity was also stimulated by halide ions, apparently through a parallel conductance pathway. Under a variety of sensitive test conditions, the reverse enzyme reaction (i.e., incorporation of 32Pi into ATP) was not detected. We conclude that this major H+-ATPase serves to acidify the abundant prelysosomal vacuoles found in D. discoideum (Padh et al. (1989) J. Cell Biol. 108, 865-874). The finding of a vacuolar H+-ATPase in a protist suggests the ubiquity of this enzyme among the eukaryotic kingdoms.