Polymers of cell-free hemoglobin have been designed for clinical use as oxygen carriers, but limited information is available regarding their effects on vascular regulation. We tested the hypothesis that the contribution of heme oxygenase (HO) to acetylcholine-evoked dilation of pial arterioles is upregulated two days after polymeric hemoglobin transfusion. Dilator responses to acetylcholine measured by intravital microscopy in anesthetized cats were blocked by superfusion of the HO inhibitor tin protoporphyrin-IX (SnPPIX) in a group that had undergone exchange transfusion with hemoglobin two days earlier, but not in surgical sham and albumin-transfused groups. However, immunoblots from cortical brain homogenates did not reveal changes in expression of the inducible isoform HO1 or the constitutive isoform HO2 in the hemoglobin-transfused group. To test if the inhibitory effect of SnPPIX was present acutely after hemoglobin transfusion, responses were measured within an hour of completing the exchange transfusion. In control and albumin-transfused groups, acetylcholine responses were unaffected by SnPPIX, but were blocked by addition of the nitric oxide synthase inhibitor N-nitro-L-arginine (L-NNA) to the superfusate. In hemoglobin-transfused groups, the acetylcholine response was blocked by either SnPPIX or L-NNA alone. The effect of another HO inhibitor, chromium mesoporphyrin (CrMP), was tested on ADP, another endothelial-dependent dilator, in anesthetized rats. Pial arteriolar dilation to ADP was unaffected by CrMP in controls, but was attenuated 62% by CrMP in rats transfused with hemoglobin. It is concluded 1) that polymeric hemoglobin transfusion acutely upregulates the contribution of HO to acetylcholine-induced dilation of pial arterioles in cats, 2) that this upregulation persists two days after transfusion when 95% of the hemoglobin is cleared from the circulation, and 3) that this acute upregulation of HO signaling is ubiquitous in that similar effects were observed with a different endothelial-dependent agonist (ADP) in a another species (rat).