Vasoconstriction is a major adverse effect of first and second generation hemoglobin-based oxygen carriers (HBOCs) that hinders their development as blood substitute. However, intravenous infusion of HBOC-201 (second generation) to patients induces significant pulmonary hypertension without significant coronary vasoconstriction. We compared contractile responses of isolated bovine pulmonary and coronary arterial strips to HBOC-201 and HBOC-205LL.LT.MW600 (third generation), polymerized bovine hemoglobins of different molecular weight, and their attenuation by nitroglycerin, sodium nitroprusside (SNP), and sodium nitrite. Pulmonary arteries developed negligible basal tone, but exhibited HBOC-dependent amplification of phenylephrine-induced contractions. In contrast, coronary arteries developed significant basal tone, and exhibited HBOC-dependent constant force increment to serotonin-induced contractions. Therefore, relative to basal tone, HBOC-induced contractions were greater in pulmonary than coronary arteries. Furthermore, HBOC-205LL.LT.MW600 appeared to be less vasoactive than HBOC-201. Unexpectedly, pulmonary and coronary arteries exhibited differential sensitivities to nitrovasodilators in parallel with their differential sensitivities to HBOC. However, SNP and sodium nitrite induced significant methemoglobin formation from HBOC, whereas nitroglycerin did not. These results suggest that phenotypic differences between pulmonary and coronary vascular smooth muscle cells could explain the differential hypertensive effects of HBOC on pulmonary and coronary circulation in patients. Among the three nitrovasodilators investigated, nitroglycerin appears to be the most promising candidate for attenuating HBOC-induced pulmonary hypertension in older HBOCs.