Regulatory mechanisms of erythrocyte glycolysis and 2,3-diphosphoglycerate (2,3DPG) metabolism under hypoxia were studied in rats exposed to a simulated altitude of 18,000 ft (5,486 m) for 5 d. Changes in erythrocyte glycolytic intermediates were determined by enzymatic analysis. Marked alterations of glycolytic intermediates were found during 1 d of exposure which were quite different from those observed during exposure for 2, 3, and 5 d. Alterations of intermediates seem to be highly correlated with blood pH changes; however, pH alone cannot explain the overall changes in intermediates. Results suggested that overall intermediate changes are the results of the combined effect of alterations of cellular pH and hemoglobin desaturation. Increased 2,3DPG at initial stages of exposure (within 1 d) may be caused mainly by the increased cellular pH; sustained elevation of 2,3DPG at later stages could be attributed to the relief of product inhibition of diphosphoglycerate mutase by deoxygenation.