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 CO₂ 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 (P₅₀) 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 P₅₀.