Allosteric regulation by cytosolic Ca2+ of Na(+)/Ca2+ exchange activity in the Ca2+ efflux mode has received little attention because it has been technically difficult to distinguish between the roles of Ca2+ as allosteric activator and transport substrate. In this study, we used transfected Chinese hamster ovary cells to compare the Ca2+ efflux activities in nontransfected cells and in cells expressing either the wild-type exchanger or a mutant, Delta(241-680), that operates constitutively; i.e., its activity does not require allosteric Ca2+ activation. Expression of the wild-type exchanger did not significantly lower the cytosolic Ca2+ concentration ([Ca2+](i)) compared with nontransfected cells. During Ca2+ entry through store-operated Ca2+ channels, Ca2+ efflux by the wild-type exchanger became evident only after [Ca2+](i) approached 100-200 nM. A subsequent decline in [Ca2+](i) was observed, suggesting that the activation process was time dependent. In contrast, Ca2+ efflux activity was evident under all experimental conditions in cells expressing the constitutive exchanger mutant. After transient exposure to elevated [Ca2+](i), the wild-type exchanger behaved similarly to the constitutive mutant for tens of seconds after [Ca2+](i) had returned to resting levels. We conclude that Ca2+ efflux activity by the wild-type exchanger is allosterically activated by Ca2+, perhaps in a time-dependent manner, and that the activated state is briefly retained after the return of [Ca2+](i) to resting levels.