The importance of hemoglobin-oxygen affinity (HOA) in affecting skeletal muscle oxygen consumption (VO2) was reevaluated using an isolated canine gracilis muscle. HOA of the blood [normal O2 half-saturation pressure of hemoglobin (P50) = 30 Torr] was increased by refrigerated storage (P50 = 22 Torr), incubation in sodium metabisulfite (P50 = 24 Torr), or in sodium cyanate (P50 = 14 Torr). Stored blood caused a significant fall in VO2 to 80% of control, with no change in venous O2 partial pressure (PvO2), substantiating previous studies. However, in contrast, blood incubated in sodium metabisulfite or sodium cyanate resulted in no impairment of VO2, with a fall in PvO2 in the latter case indicating that a critical PvO2 did not cause the reduction in VO2 with stored blood. To substantiate further the lack of existence of a critical PO2, fresh and increased HOA blood was perfused at constant flow rates and varying arterial oxygen saturations. Stored blood showed a marked reduction in VO2 as compared with normal blood over a wide range of saturations. However, carbamylated blood VO2 was identical to fresh blood VO2 values. The data suggest that the position of the oxygen dissociation curve may not be as important as originally thought in determining skeletal muscle oxygen delivery. The drop in VO2 caused by perfusion with stored blood is due to some other factor unrelated to HOA.