The ATP dependence of the Na-Ca exchanger was investigated in isolated adult rat heart cells to evaluate the extent to which ATP depletion after a period of ischemia plus reperfusion in whole hearts could limit calcium uptake by Na-Ca exchange. A standard state for measurement of Na-Ca exchange activity that could be used with cells depleted of ATP to different degrees was defined. This was a state of zero sarcolemmal gradient for sodium, potassium, and pH and was achieved by incubation of the cells for 5 minutes with EDTA, EGTA, ouabain, and nigericin. Heterogeneity of cell ATP levels was minimized by using a protocol of total ATP depletion by incubation under conditions similar to ischemia, followed by reoxygenation to give partial restoration of ATP levels. No ATP was regenerated when cells were reoxygenated in the presence of rotenone, and such cells showed a very low rate of calcium uptake. Without rotenone, cells showed an almost complete restoration of Na-Ca exchange activity, in spite of a restoration of ATP levels to only one third of control values. Thus, the dependence of calcium uptake on ATP was highly nonlinear under these conditions. The calculatedKmfor ATP was no more than 10% of normal ATP levels. We conclude that ATP depletion after ischemia plus reperfusion is unlikely to limit the rate of calcium uptake through Na-Ca exchange in the whole heart if at least one quarter of the ATP is restored. In addition, we measured the apparent ATP dependence of calcium uptake by Na-Ca exchange in cells under conditions in which we previously had concluded that cell ATP distributions were very heterogeneous: when cells undergo contracture during incubation with oligomycin and without glucose. A linear relation between calcium uptake rate and ATP was observed at all ATP levels. This can be understood if cells in contracture that are incubated with oligomycin cannot take up calcium because of low ATP, whereas rod-shaped cells are able to retain a full uptake capability. This result further supports our conclusion that the ATP level declines catastrophically to near zero in these oligomycin-incubated cells just before contracture.