High concentrations of cytosolic Na⁺ ions induce the time-dependent formation of an inactive state of the Na⁺/Ca²⁺ exchanger (NCX), a process known as Na⁺-dependent inactivation. NCX activity was measured as Ca²⁺ uptake in fura-2 loaded Chinese hamster ovary (CHO) cells expressing either the wild-type (WT) NCX, or mutants that are either hypersensitive (F223E) or resistant (K229Q) to Na⁺-dependent inactivation. As expected, (a) Na⁺-dependent inactivation was promoted by high cytosolic [Na⁺], (b) the F223E mutant was more susceptible to inactivation than the WT exchanger while the K229Q mutant was resistant, and (c) inactivation was enhanced by cytosolic acidification. However, in contrast to expectations from excised patch studies, (a) the WT exchanger was resistant to Na⁺-dependent inactivation unless the cytosolic pH was reduced, (b) reducing cellular phosphatidylinositol-4,5-bisphosphate levels did not induce Na⁺-dependent inactivation in the WT exchanger and Na⁺-dependent inactivation (c) did not increase the K_h for allosteric Ca²⁺ activation, (d) was not reversed by high concentrations of cytosolic Ca²⁺, and (e) was partially, but transiently reversed by increasing the extracellular Ca²⁺ concentration. Thus, Na⁺-dependent inactivation of NCX expressed in CHO cells differs in several respects from the inactivation process measured in excised patches. The refractoriness of the WT exchanger to Na⁺-dependent inactivation suggests that this type of inactivation is unlikely to be a strong regulator of exchange activity under physiological conditions, but would probably act to inhibit NCX-mediated Ca²⁺ influx during ischemia.