The vacuolar membrane of plant cells is characterized by two proton pumps: the vacuolar H⁺‐ATPase (V‐ATPase; EC 3.6.1.3) and the vacuolar H⁺‐PPase (V‐PPase; EC 3.6.1.1). Recently, Du Pont and Morrissey reported that Ca²⁺ stimulates hydrolytic activity of purified V‐ATPase (Arch. Biochim. Biophys., 1992. 294: 341–346). Since this effect may be due to degradation during purification further investigation of Ca²⁺ regulation of native V‐ATPase was done. However, native tonoplast membranes contain a Ca²⁺/H⁺ antiport activity, which interferes with effects of calcium ions on proton transport activity of vacuolar ATPase. Therefore, the effects of anti‐calmodulin drugs (W‐7, W‐5, calmidazolium), and calcium channel antagonists (Verapamil, Diltiazem) on proton transport activities of the vacuolar‐type H⁺‐ATPase and H⁺‐PPase in tonoplast enriched membrane vesicle preparations from roots of Zea mays L. were studied. The concentrations for half maximal inhibition of vacuolar H⁺‐ATPase (H⁺‐PPase) were: 71 (191) μM W‐7, 470 (> 800) μM W‐5, 26 (24) μM calmidazolium (= compound R 24571). 398 (700) μM Verapamil, and 500 (1 330) μM Diltiazem. Estimation of Hill coefficients (n_H) for the inhibition by Verapamil showed a further difference between the two vacuolar proton pumps (H⁺‐ATPase, n_H= 2.02; H⁺‐PPase, n_n= 0.96). The data indicate that the vacuolar H⁺‐ATPase itself is affected by these chemicals. It is suggested that some biological activities of W‐7, W‐5, Verapamil, and Diltiazem are due to their effects on proton translocation by the vacuolar‐type H⁺‐ATPase.