The effect of pH on the rate of Ca2+ efflux mediated by the Ca2+ adenosinetriphosphatase (ATPase) was measured. The cations Ca2+, Mg2+, and K+ decrease the rate of Ca2+ efflux at pH 7.5 but not at pH 6.0. The effect of pH on the affinity to Ca2+ during Ca2+ efflux was found to be similar to the pH dependence of the high-affinity Ca2+ binding sites of the ATPase. The inhibitory activity of cations was significantly increased by a rise in pH, whereas acidification amplified the magnitude of an efflux component insensitive to cations. Acidification of the assay medium allows efflux of Ca2+ through the Ca2+ pump, even in the presence of high concentrations of monovalent and divalent cations. The efflux rate was severalfold increased by addition of the hydrophobic drugs trifluoperazine, dibucaine, and imipramine. At neutral pH, the Ca2+ efflux induced by trifluoperazine was antagonized by the cations Ca2+, Mg2+, and K+ and by thapsigargin, a highly specific inhibitor of the Ca2+ pump. In contrast to that observed at neutral pH, the cations did not antagonize the effect of trifluoperazine on Ca2+ efflux at acid pH. It is concluded that H+ produces functional alterations in ATPase domains involved in Ca2+ efflux.