The plasma membrane of spinach leaves was purified by two phase partitioning and the H⁺pumping activity associated to the purified plasma membrane was characterized. The pH optimum was 6.7 similar to that of the ATPase activity and the substrat specificity was more obvious for ATP with a Kmapp of 0.697. The H⁺pumping activity was more or less inhibited by different ATPase inhibitors, and especially by VO₄ with I₅₀ of 160 μM. The SDS-PAGE of the purified plasma membrane proteins revealed the presence of a polypeptide of about 100 kDa which crossreacted with the polyclonal antibodies against the plasma membrane ATPase of Arabidopsis thaliana.