Magnesium deficiency lowers endurance capacity of untrained rats by an as yet unknown mechanism. We tested the hypothesis that low plasma and blood Mg concentrations would reduce blood 2,3-diphosphoglycerate (2,3-DPG) concentrations, thus diminishing the oxygen delivery system of the Mg-deficient rat. Untrained male Osborne-Mendel rats (95 g) were studied at rest or following exhaustive exercise after three weeks of dietary treatment (400 or 100 micrograms Mg/g diet; deionized or high Mg water [greater than 85 micrograms Mg/ml]). Magnesium deficiency resulted in lowered plasma and red blood cell (RBC) Mg concentrations and reduced endurance capacity compared with controls. Magnesium supplementation normalized these parameters. 2,3-DPG was elevated by 10% in rats acutely exercised compared with sedentary rats; however, 2,3-DPG levels were not affected by dietary Mg intake. Acute exercise tended to increase erythrocyte Mg concentrations in control rats and lower erythrocyte Mg levels in deficient rats. In conclusion, the reduced endurance capacity of Mg-deficient rats is not due to impaired 2,3-DPG production.