Experiments were performed to determine the effect of ouabain on the release of relaxing factor(s) from cultured endothelial cells, and its action on the effect of the relaxing factor(s) on arterial smooth muscle. A column of porcine aortic endothelial cells grown on microcarrier beads in suspension culture was perfused with modified Krebs-Ringer bicarbonate solution. The release of relaxing factor(s) by the endothelial cells was detected under bioassay conditions by measuring the relaxing activity of the perfusate overflowing a ring of canine coronary artery (without endothelium) contracted with prostaglandin F2α. Incubation of the endothelial cells with ouabain did not affect the relaxation of the bioassay ring under basal conditions or upon stimulation of the endothelial cells with ADP but impaired the relaxation Induced by bradykinin or the calcium ionophore A23187. Incubation of the bioassay ring with ouabain reduced the relaxation under basal conditions as well as the relaxation induced by ADP but did not affect the relaxation observed upon stimulation with bradykinin and A23187 and the endothelium- independent relaxations induced by nitric oxide. These experiments suggest that cultured porcine aortic endothelial cells release two endothelium-derived relaxing factors; one is released under basal conditions and upon stimulation with adenosine diphosphate and the other (which presumably is nitric oxide) upon stimulation with bradykinin and the calcium ionophore A23187.