Hemoglobin (Hb)-based artificial oxygen carriers are supposed to induce vasoconstriction through the inactivation of endothelium-derived relaxing factor (EDRF). We examined the vasoconstrictive activity of acellular Hb and cellular Hb solutions in rabbit aortic strips. Unmodified Hb, pyridoxalated Hb, bovine unmodified Hb, haptoglobin-Hb complex (Hp-Hb), and polyoxyethylene glycol-conjugated Hb (PEG-Hb) were used as acellular Hbs having different molecular masses. Cellular Hbs included liposome-encapsulated Hb and red blood cells (RBC). In the first experiment, Hb (10 ng/ml to 1 mg/ml) was cumulatively added to the tissues in which steady-state relaxation was evoked by acetylcholine (ACh) after precontraction induced by phenylephrine. Although all Hb solutions induced a dose-dependent reversal of ACh-induced relaxation, the most potent vasoconstrictive effect was noted with acellular Hbs, and their contractile activities were almost the same independent of molecular mass. On the other hand, liposome-Hb and RBC showed reduced potencies in this order. These results indicate the importance of cellularity as the major factor determining Hb-related EDRF inactivation. In another experiment, the tissues were exposed to Hb at 0.01, 0.1, or 1 mg/ml for 30 min and ACh-induced relaxation was recorded after the complete removal of Hb in an organ bath chamber. Exposure to unmodified Hb at> 0.1 mg/ml concentrations significantly reduced the ACh-induced relaxation, whereas the relaxation was not affected by PEG-Hb, Hp-Hb, liposome-Hb, or RBC. These results suggest that unmodified Hb might be persistently associated with tissues and thereby inhibit ACh-induced relaxation. From these findings, we propose two attributes of Hb-related inhibition of endothelium-dependent relaxation: Acellular Hbs inhibit EDRF more efficiently in the luminal space than cellular Hbs, and unmodified Hb can also inhibit it adluminally and/or adventitially.