Plasma membrane vesicles were purified from tobacco callii and the modulation of H⁺-ATPase by detergents was investigated. The nonionic detergent Brij 58 not only activated ATP hydrolysis (2-fold) but also proton pumping (more than 4-fold). Triton X-100, within a more limited concentration range, produced a similar effect. The simultaneous activation of ATP hydrolysis and proton pumping is not compatible with current interpretations of effects of nonionic detergents on the H⁺-ATPase based on latency of the enzyme and opening of vesicles.