A coronary vasoconstrictor effect of human stroma-free hemoglobin (SFH) was identified in isolated rabbit hearts perfused with Krebs-Henseleit buffer or whole rabbit blood at a constant coronary flow rate. In buffer-perfused hearts, SFH in concentrations of 5 to 200 mg/dl produced dose-related increases of coronary perfusion pressure. At a concentration of 150 mg/dl, SFH, equilibrated with CO to form carboxyhemoglobin, caused an increase in perfusion pressure (55 +/- 7 mmHg), similar to that observed with oxyhemoglobin (57 +/- 6 mmHg); addition of potassium ferricyanide to form methemoglobin reduced the increase of perfusion pressure to 34 +/- 5 mmHg (P less than 0.05). The vasoconstrictor activity could not be eliminated by dialyzing against the perfusion buffer. Human SFH prepared by different methods had similar vasoconstrictor activity. Rabbit SFH and human SFH were equi-effective in the rabbit heart. Less constrictor activity of SFH was evident in rat and guinea pig heart. Polymerized, pyridoxalated SFH had greatly reduced constrictor effect compared with unmodified or pyridoxalated tetramer SFH. In blood-perfused hearts, increasing plasma hemoglobin to 1.6 +/- 0.1 g/dl, without changing total hemoglobin or arterial O2 content, increased coronary perfusion pressure by 36 +/- 13 mmHg (P less than 0.05). We conclude that stroma-free hemoglobin solutions exert a coronary vasoconstrictor effect that is unrelated to O2 delivery.