Influence of altered blood oxygen affinity on maximal performance ability was evaluated in trained rats exercising to exhaustion in a graded treadmill test. Modification of blood oxygen affinity was achieved both by 2,3-diphosphoglycerate depletion, accomplished by exposure of animals to CO(2) and by exchange transfusion with blood exposed to bisulfite or stored in acid citrate dextrose, and by carbamylation of hemoglobin, produced by exchange transfusion of blood incubated with potassium cyanate. A decrease in oxygen tension at half-saturation of hemoglobin (P(50)) from 36 to 23 mm Hg produced a decrease in resting central venous oxygen pressure of about 12 mm Hg. During exercise it caused an average decrease in work performance of about 10%, which was equivalent to that performance decrement caused by a decrease in hemoglobin concentration of approximately 10%. When superimposed on anemia, this change in blood oxygen affinity again caused a similar decrease in performance over and above that due to anemia alone. A marked rightward shift of the in vivo oxygen dissociation curve during severe exercise may have compensated for the reduced in vitro P(50).