A B S T R A C T Normal red cells were incubated in the absence of glucose to develop a system in which total adenosine triphosphate (ATP) turnover could be assessed. After 1 hr, the triose pool had been completely consumed. Thereafter, the metabolism of 2,3-diphosphoglycerate (DPG) to pyruvate and lactate was the sole significant source of ATP synthesis.1-'M CuC12, which did not enter the cells, diminished ATP utilization by more than 50%. This could be only partially attributed to the inhibition by copper of residual acylation and cation pumping, which were already reduced by glucose depletion. Other membrane enzymes, which presumably function in the maintenance of membrane integrity, must, therefore, use a significant portion of erythrocyte ATP.The behavior of glucose-depleted red cells with respect to cation transport was complex. The addition of ouabain did not decrease ATP utilization in these red cells. Ouabain inhibitable potassium influx was nearly normal after triose depletion, but total potassium influx was decreased. In contrast, the ouabain inhibitable sodium efflux was markedly reduced after triose depletion, although the concentration of ATP was 70% of normal. The dissociation of monovalent cation pumping suggests that the energy for active sodium transport is derived from a specific source (such as the ATP produced by the phosphoglycerate kinase reaction) distinct from that for potassium transport.