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The affinity of hemoglobin for oxygen may increase significantly in subjects who are hypophosphatemic and alkalotic. We studied the organic phosphate content and oxygen binding by hemoglobin of red cells in subjects undergoing hemodialysis, during which time a decrease in plasma inorganic phosphate and an increase in blood pH may occur. Red cell 2,3-DPG was not correlated with plasma inorganic phosphorus, whereas red cell ATP was highly correlated with plasma inorganic phosphorus when analyses were made on predialysis samples. Predialysis red cell inorganic phosphorus was highly correlated with plasma inorganic phosphorus, supporting the concept that intraerythrocytic inorganic phosphorus is maintained by a gradient from plasma to cell. Plasma inorganic phosphorus decreased by 45% during the period of hemodialysis, whereas red cell inorganic phosphorus did not change. Red cell 2,3-DPG, ATP, and oxygen binding by hemoglobin at standard conditions of temperature, pH, and pCO2 were not altered after 6 hr of hemodialysis. Plasma pH and base excess increased during dialysis. The increase in base excess, an estimate of the non-pH-dependent effect of CO2 on oxygen binding by hemoglobin, counterbalanced a portion of the effect of elevated pH on hemoglobin— oxygen affinity under in vivo conditions. Hence, only a slight increase in oxygen binding by hemoglobin occurred. Moreover, late dialysis symptoms were not associated with the degree of alkalosis or with the extent of change in hemoglobin’s affinity for oxygen. Red cell 2,3-DPG content was lower and hemoglobin’s affinity for oxygen was higher in subjects with chronic renal disease than in nonazotemic subjects with similar hemoglobin deficits. Moreover, increased red cell ATP in chronic renal disease patients did not influence oxygen binding by hemoglobin.