The transport-competent plasma membrane vesicles were obtained using a modified homogenization medium by the two-phase partitioning method from soybean hypocotyls. It was noted that freeze-thaw treatment could increase the amount of inside out vesicles.Further experiments showed that the fluorescence quenching caused by adding of Mg^2＋-ATP could be dissipated by the proton ionophore CCCP, and inhibited by the proton transport inhibitor DCCD; it was also found that the plasma membrane H^＋-ATPase specific inhibitor vanadate could quench the fluorescence completely. The fluorescene quenching was Mg²⁺-dependent, K^＋-stimulated, with an optimum pH of 6.5. All the above results indicated that the fluorescence quenching was caused by the transport of protons by the plasma membrane H^＋-ATPase. It was also suggested that the maintaining of suitable conformation of the plasma membrane H^＋-ATPase,and the tightness of plasma membrane vescials are the two key factors for the proton transport determination.