Calcium accumulation in intact human erythrocytes is enhanced by incubation in hypertonic solutions. Hypertonicity produces an increased permeability of the membrane to calcium that is time-dependent and occurs in the presence or absence of calcium. When hypertonically treated cells are incubated for more than 30 min in 300 mosmol/kg solutions the permeability of the membrane to calcium returns to basal values. Oligomycin inhibits the effect of hypertonicity on calcium uptake. The inhibitory action of oligomycin diminishes as the external sodium increases and can only be observed when the external concentration of potassium is at or below 3 mM. Low intracellular sodium and high intracellular potassium concentrations increase the uptake of calcium. It is concluded for human erythrocytes that 1) the increased permeability of the membrane to calcium produced by hypertonicity is a time-dependent, reversible phenomenon and is independent of calcium, 2) the increase in intracellular potassium concentration associated with hypertonic exposure is an important factor contributing to this response, and 3) interactions between calcium and components of the sodium-potassium transport system may account for the enhanced uptake of calcium produced by hypertonicity.