The vasoactive effects of media obtained from bovine aortic endothelial cells (EC) in culture were directly tested on isolated rings of the porcine left anterior descending coronary artery. Increasing concentrations of EC-conditioned culture media resulted in progressive dose-dependent increments in isometric tension in porcine, bovine, and canine coronary arteries; the response did not require an intact endothelium. Control (nonconditioned) media and that conditioned by fibroblasts or vascular smooth muscle cells in culture had negligible effects on vessel tone. The vasoconstriction required extracellular Ca2+ and was unaffected by inhibitors of cyclooxygenase and lipoxygenase or by antagonists to the alpha- or beta-adrenergic, serotonergic, histaminergic, or cholinergic receptor systems. Calibrated gel filtration of the media indicated a molecular weight of 8,500 for the vasoactive factor; treatment of the EC-conditioned media with either sodium dodecyl sulfate, trypsin, alkali, or with acid hydrolysis completely abolished the vasoconstrictive effect. These findings and others now provide evidence for the existence of an EC-derived polypeptide vasoconstrictor that may be important in the regulation of vascular smooth muscle contractility.