An acid slab gel electrophoresis method of high-resolving power allows detection of a phosphorylated form in the purified ATPase of the yeast Schizosaccharomyces pombe and identification of this catalytic intermediate among the different phosphopeptides of a plasma membrane preparation. At a maximum steady state rate of MgATP hydrolysis by the membrane-bound ATPase, 20 to 40% of the ATPase subunits of 100,000 daltons are in a phosphorylated form, while only 0.8% of the subunits of the purified ATPase are phosphorylated under the same conditions. The phosphorylated intermediate reaches the steady state level in less than 2 s and rapidly turns over. The phosphorylated substance is cleaved by hydroxylamine and is relatively stable in acids but is readily hydrolyzed in alkaline or in acid alcoholic media. These results suggest that the intermediate is an acylphosphate. The phosphorylation reaction has an apparent Km value of 3.0 mM MgATP for the plasma membrane-bound ATPase and 0.6 mM MgATP for the purified ATPase. Plasma membranes contain several other minor phosphorylated components whose kinetic behavior is typical of phosphorylation by protein kinase. Artifactual production of two forms of the ATPase by phenylmethanesulfonyl fluoride-sensitive proteases liberated during cell disruption is also demonstrated.