We assessed hemoglobin affinity for oxygen (standard P50: pH 7.40, PCO2 40 Torr, temperature 37 degrees C) in 3 series of studies of 16 male subjects who performed exhaustive treadmill exercise of 10 min duration. Standard P50 was significantly elevated (0.7--2.5 Torr) during the last minute of and for 30 min following exercise. Exercise while carboxyhemoglobin (HbCO) was acutely elevated to 5% did not affect std P50 any differently than exercise alone. 2,3-Diphosphoglycerate (2,3-DPG) was significantly elevated after exercise, but not during or 30 min postexercise, and it correlated significantly with P50 in 2 studies (r, 0.43--0.68). ATP (mumol/100 ml) was significantly elevated only 3 min postexercise. Serum [Na+], [K+], [Cl-], osmolarity, and hemoglobin (Hb) concentration were significantly elevated during exercise with [Na+] osmolarity, and [Hb] remaining significantly elevated postexercise. There was no significant correlation between std P50 and any of these variables. Multiple correlation regression analysis between P50 and 2,3-DPG, ATP, and [Na+] yields a coefficient of 0.86. We conclude this type of exercise is unique in causing a small change in std P50 that appears to be mediated by several factors.