The association of K(+)-stimulated, Mg(2+)-dependent ATPase activity with plasma membranes from higher plants has been used as a marker for the isolation and purification of a plasma membrane-enriched fraction from cauliflower (Brassica oleraceae L.) buds. Plasma membranes were isolated by differential centrifugation followed by density gradient centrifugation of the microsomal fraction. The degree of purity of plasma membranes was determined by increased sensitivity of Mg(2+)-ATPase activity to stimulation by K(+) and by assay of approximate marker enzymes. In the purified plasma membrane fraction, Mg(2+)-ATPase activity was stimulated up to 700% by addition of K(+). Other monovalent cations also markedly stimulated the enzyme, but only in the presence of the divalent cation Mg(2+). Ca(2+) was inhibitory to enzyme activity. ATPase was the preferred substrate for hydrolysis, there being little hydrolysis in the presence of ADP, GTP, or p-nitrophenylphosphate. Monovalent cation-stimulated activity was optimum at alkaline pH. Enzyme activity was inhibited nearly 100% by AgNO(3) and about 40% by diethylstilbestrol.