The release of ATP from red blood cells (RBC) in response to low O2 levels is linked to ATP production and the oxygenation state of hemoglobin. Because O2 is unloaded from the RBC, the concentration of deoxygenated hemoglobin increases, displacing phosphofructokinase from the cytoplasmic domain of band 3. We hypothesize that the ATP molecules produced through this glycolytic stimulation at the membrane surface result in the release of ATP from the RBC. Rat whole blood exposed to 5 min of low PO2 in vitro increased plasma [ATP] by 1.0 miccroM (+45%). This increase was reduced to 0.1 microM (+12%, P < 0.05) after citrate incubation and reversed after fluoride treatment (both glycolytic inhibitors) by -0.2 microM (-23%, P < 0.05). Plasma [ATP] of control RBC decreased -0.3 microM (-12%) when 8% CO (P < 0.05) was added to the chamber. Because CO and O2 bind competitively to heme, these results support our hypothesis that the release of ATP from RBC is linked to ATP production through the oxygenation state of the hemoglobin molecule.