Plasma membrane‐enriched vesicles were isolated from maize roots, and pH‐induced changes of the physical state of the plasma membrane were monitored with a fluorescence probe, TMA‐DPH. At physiological temperatures, the membrane's microviscosity increased upon acidification of the vesicles’ suspending medium. It seemed that membrane protonation led to a reorganization of maize root plasma membranes. ATP‐dependent proton pumping activity across the vesicular membrane was assayed by quinacrine fluorescence quenching. By comparing the steady‐state level of pumping activity, the proton pumping activity at pH 6.5 was about three times that determined at pH 5.5. Proton pumping apparently originated in a plasma‐membrane bound Mg2+‐ATPase.