To study the mechanism by which cell-free hemoglobin preparations may alter coronary vascular reactivity, we investigated the effect of human hemoglobin cross-linked between alpha chains with bis(3,5-dibromosalicyl)fumarate (alpha alpha Hb) on the vasomotor response to acetylcholine (ACh) in isolated perfused rabbit hearts. Dose-response curves were generated by monitoring the increase in coronary pressure during serial addition of 0.2-10 microM ACh before, during and after 20 min infusion of three test solutions: a) 0.1 g/dl alpha alpha Hb (62 microM heme); b) 0.1 g/dl alpha alpha Hb plus 60 microM deferoxamine (DFO); c) 50 microM NG-nitro-L-arginine methyl ester (L-NAME), a specific inhibitor of nitric oxide (NO) synthase. We found that the sensitivity to ACh-induced vasoconstriction was significantly potentiated in the presence of alpha alpha Hb and L-NAME. In addition, this response was only partially reversed after removal of alpha alpha Hb, except when DFO was simultaneously infused with the alpha alpha Hb solution. These findings are consistent with the idea that both NO binding to hemoglobin and iron-mediated oxygen free radical generation contribute to an altered coronary vasomotor responsiveness induced by cell-free hemoglobin.