The ATPase of the plasma membrane isolated from the yeast Schizosaccharomyces pombe catalyses a medium Pi in equilibrium H2O exchange in the presence of Mg2+ and in the absence of ATP and ADP. (formula, see text) The Pi in the E.Pi species tumbles in the active site so that each of its oxygens has an equal probability of exchange with water. The partition coefficient (Pc = k2/k2 + k-1) is 0.45. The total rate of oxygen exchange, Vex, representing the rate of incorporation of water oxygens occurring during hydrolysis of E--P into E.Pi (Vex = k-2[E--P]) is dependent on the [Pi] with an apparent Km of 177 mM, reflecting the very low affinity of the enzyme for Pi. The maximal exchange rate is 6.7 micrograms atoms of oxygen X min-1 X mg-1 of protein. The individual kinetic constants are evaluated: k2 = 3.4 X 10(3) min-1, k-2 = 5.50 X 10(5) min-1 and k-1 = 4.11 X 10(3) min-1. Under conditions of uncoupled transport, the hydrolysis of E--P is exergonic as [E.Pi]/[E--P] = k-2/k2 = 164. During hydrolysis of ATP, the rate of medium Pi in equilibrium H2O exchange activity as well as the extent of phosphorylation of the enzyme from Pi are markedly stimulated: 7.9 and 5.3 times, respectively, whereas the Pc is not modified. These data are most simply interpretated by the existence of two isomeric forms of the enzyme; one is specific for binding ATP and the other for binding Pi. The Pc for intermediate Pi in equilibrium H2O exchange, when the E--P species is formed from cleavage of [gamma-18O]ATP, is the same as for medium exchange, indicating that the same exchange pathway operates under both conditions. Varying the [ATP] had very little effect on the Pc, indicating little or no cooperativity between different catalytic sites under the conditions used in this study.