Vascular responsiveness to various vasoactive substances after exchange transfusion with a modified hemoglobin (pyridoxalated hemoglobin polyxyethylene conjugate; PHP) was examined in anesthetized rats. The hindlimb region of the rat was perfused through the abdominal aorta with autologous blood at a constant flow rate, using a perfusion pump. Perfusion pressure (PP), systemic blood pressure (SBP), and heart rate were measured. Exchange transfusion with a 6% PHP solution (20 ml/kg) was performed and effects of intra-arterial injections of norepinephrine (NE), angiotensin-II (A-II), serotonin (5-HT), acetylcholine (ACh), and nitroglycerin (NG) on PP and SBP were examined. The NE and A-II induced a dose-dependent increase in PP, while ACh and NG induced a decrease in PP. There was no difference in vascular responsiveness to ACh, NG, A-II, and 5-HT before and after exchange transfusion with PHP solution, but NE-induced responses were augmented after exchange transfusion with PHP. Results suggest that the presence of PHP moiety in circulation would not alter vascular responsiveness to ACh, NG, A-II, and 5-HT in a significant manner.