Recent reports indicate that vacuolar-type proton ATPases from chicken osteoclasts (Chatterjee et al. (1992) Proc. Natl. Acad. Sci. USA 89, 6257-6261), yeast vacuoles and chromaffin granules (Beltran and Nelson (1992) Acta Physiol. Scand. Suppl. 607, 41-47) can be inhibited by vanadate, albeit at a concentration much higher than that required to inhibit P-type ATPases. We have characterized the mechanism by which vanadate inhibits vacuolar-type ATPase-mediated proton transport by chicken kidney microsomes. The initial rate of proton transport is somewhat less sensitive to vanadate than the total acidification, with IC50 values of 1.58 mM and 0.78 mM vanadate, respectively. The inhibition of both the initial rate and total acidification is noncompetitive with respect to ATP. The inhibition is abolished when ADP is removed by an ATP-regenerating system, and the addition of exogenous ADP increases the vanadate inhibition of proton transport in a synergistic manner, thus demonstrating that inhibition by vanadate is dependent on the presence of ADP and explaining the lower effect of vanadate on the initial rate of acidification. Phosphate protects proton transport activity from inhibition by vanadate. These effects of ADP and phosphate suggest that inhibition by vanadate may involve the formation of a complex with ADP at a nucleotide binding site, possibly at the catalytic site of the enzyme.