Incubation of oat root plasma membrane vesicles in the presence of ATP with trypsin or chymotrypsin increased the rate of ATP hydrolysis and ATP-dependent proton pumping by the plasma membrane H⁺-ATPase. Proton pumping was stimulated more than 200%, whereas ATP hydrolytic activity was stimulated about 30%. The K(m)(ATP) for both proton pumping and ATP hydrolysis was lowered from about 0.3 mM to below 0.1 mM. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of trypsin-treated plasma membranes revealed a decrease in a 100-kDa band and the appearance of a 93-kDa band. Western blot analysis using antibodies against the H⁺-ATPase showed that both of these bands represented the H⁺-ATPase and suggested that a 7-kDa segment was released. Extensive treatment with cardoxypeptidase A also activated the H⁺-ATPase indicating that the 7-kDa segment originated from the C terminus.