Diaspirin-cross-linked hemoglobin (DCLHb; Baxter Healthcare Corp) has potential for clinical use as a hemoglobin-based oxygen-carrying solution. We have previously shown that DCLHb administration is associated with a self-limiting increase in mean arterial pressure (MAP). Based on in vitro studies with other hemoglobin solutions, this vasopressor effect is thought to be mediated at least in part by the release or inhibition (or both) of endothelium-derived vasoactive substances. The purpose of our studies was to determine the role of endothelin (ET), a potent vasoconstrictor peptide, and nitric oxide, a vasodilator, in mediating the pressor effect of DCLHb in conscious rats. Intravenous administration of DCLHb has been shown to elicit an immediate increase in MAP that peaks within 30 minutes and returns to baseline by 300 minutes in a dose-dependent fashion. Phosphoramidon, an inhibitor of proendothelin conversion to ET, attenuated the elevation of MAP when administered before DCLHb. Administration of cyanomet DCLHb, a DCLHb molecule that is unable to interact with NO, was not associated with an elevation of MAP. L-arginine, the substrate for NO synthesis, and nitroglycerin, an NO donor, significantly reduced MAP when infused 15 minutes after DCLHb administration. Based on these findings, we conclude that the DCLHb-induced elevation of MAP in vivo is mediated at least in part by ET and the inhibition of NO. Although these data support earlier reports of hemoglobin binding NO, this is the first report of the pressor response to hemoglobin being attenuated by an agent that blocks the conversion of proendothelin to ET.