Hypothermia decreases the arterial PO₂ at which hemoglobin is 50% saturated (P₅₀), increasing hemoglobin O₂-binding affinity. We used RSR13, a synthetic allosteric modifier of hemoglobin that increases P₅₀, to study the role of altered hemoglobin O₂-binding affinity in mild hypothermic neuroprotection. RSR13 (150 mg/kg iv) restored P₅₀ to normothermic values. Rats underwent 70 min of middle cerebral artery occlusion (MCAO) at 30.0, 34.0, or 37.5°C with hemoglobin saturation held at 98-100%. The 34.0°C group received RSR13 or vehicle before ischemia. After 7 days of recovery, infarct volumes were reduced in all hypothermic groups, without evidence of a detrimental effect on infarct size or neurological score as a result of P₅₀ correction. To examine for a beneficial effect of P₅₀ correction, ischemia duration was increased to 120 min in rats maintained at 34.0°C. Correction of P₅₀ by RSR13 did not alter cerebral infarct sizes or neurological scores. The decrease in P₅₀, caused by mild hypothermia, could not be associated with infarct size or neurological deficit resulting from ischemic brain hypoxia in rats.