Partial exchange transfusion with a cell-free hemoglobin (Hb) polymer during transient middle cerebral artery occlusion (MCAO) reduces infarct volume but fails to increase blood flow, as might be expected with the induced decrease in hematocrit. In ischemic brain, endothelin antagonists are known to produce vasodilation. In non-ischemic brain, pial arterioles constrict after Hb exchange transfusion, and the constriction is blocked by an inhibitor of 20-HETE synthesis. We tested the hypothesis that a 20-HETE synthesis inhibitor and an endothelin A receptor antagonist increase pial arteriolar dilation after Hb exchange transfusion during MCAO. Pial arteriolar diameter was measured in the ischemic border region of the distal MCA border region through closed cranial windows in anesthetized rats subjected to the filament model of MCAO. During 2 h of MCAO, pial arteriolar dilation gradually subsided from 37±3% to 7±5% (±SE). Compared to residual dilation at 2 hours of MCAO with vehicle superfusion (14±3%), loss of dilation was not prevented by superfusion of a 20-HETE synthesis inhibitor (21±5%), partial Hb exchange transfusion (7±5%) that decreased hematocrit to 23%, or a combination of the two (5±5%). However, loss of dilation was prevented by superfusion of an endothelin A receptor antagonist with (35±4%) or without (32±5%) Hb transfusion. Pial artery constriction during reperfusion was attenuated by HET0016 alone and by BQ610 with or without Hb transfusion. Systemic administration of the endothelin antagonist during prolonged MCAO increased blood flow in the border region. Thus, loss of pial arteriolar dilation in the ischemic border region during prolonged MCAO depends on endothelin A receptor activation, and this effect was independent of the presence of cell-free Hb polymers in the plasma. In contrast to previous work in non-ischemic brain, inhibition of oxygen-dependent 20-HETE synthesis does not significantly influence the pial arteriolar response to polymeric Hb exchange transfusion during focal ischemia.