Adenosinetriphosphatase (ATPase) activity stimulated by K+ and inhibited by Sch-28080 (SCH), omeprazole (OME), and vanadate has been measured in microsomes from mammalian renal medulla and attributed to a kidney isoform of the H(+)-K(+)-ATPase. To determine whether the H(+)-K(+)-ATPase inhibitors could also inhibit the vacuolar (V)-type H(+)-adenosinetriphosphatase (H(+)-ATPase, i.e., H+ pump) in mammalian intracellular vesicles, we examined their effects on bafilomycin-sensitive acidification in renal cortical vesicles (CEV) and medullary endocytic vesicles (MEV). Rats were injected with fluorescein isothiocyanate-labeled dextran, and labeled endosomes were enriched from kidney tissue homogenates by differential and Percoll density gradient centrifugation. In the CEV, the V-type H+ pump was inhibited 25% by SCH and 30% by OME (100 microM each). Whereas the inhibition by OME was concentration and time dependent, the inhibition by SCH was only concentration dependent. Inhibition by these compounds was similar in the presence of 50 mM K+ (in = out) and in the complete absence of K+, thus ruling out a significant involvement of H(+)-K(+)-ATPase-mediated acidification. Inhibition, however, was not observed with 10 microM SCH and OME. The sensitivity of the V-type H+ pump to 100 microM SCH and OME in CEV was confirmed by the comparable inhibitions of intravesicular acidification observed in acridine orange fluorescence quench studies and by inhibition of Pi liberation in an ATPase assay. We also found that the V-type H+ pump in isolated rat liver endosomes is sensitive to 100 microM SCH and OME to a similar degree. In the MEV, acidification was only weakly affected by 100 microM SCH and OME, thus suggesting that H(+)-ATPases in endosomes from cortical and medullary tubules are different, possibly due to a previously described selective expression of subunit isoforms. Our finding indicates the importance of using low concentrations (< 10 microM) of OME and SCH in studies of H(+)-K(+)-ATPase in nongastric tissues to avoid misinterpretation of the data due to nonspecific inhibition of V-type H(+)-ATPases.