The aim of this study was to determine if endothelial cells in the heart release substances into the coronary perfusion medium that modify the contractility of myocardial cells. To assay the effects on the contractility of cardiac muscle of fluid that has passed through the coronary vasculature, a new method has been developed based on the cascade principle used to study vascular smooth muscle function. The coronary venous effluent from an isolated perfused working heart was collected periodically, and after reoxygenation it was used as the bathing medium for trabeculae isolated from the endocardial surface of another heart. The coronary venous effluent changed the contraction of the isolated trabeculae. The amplitude and the direction of the change depended on the degree of oxygen saturation of the coronary effluent before it was reoxygenated and the rate of coronary flow at the time the effluent was collected. The response of the trabecula to the coronary effluent was substantially altered by damaging the endocardial endothelium with a 1-second exposure to 0.5% Triton X-100 in Krebs' solution. It was completely eliminated by damaging endothelial cells in both the perfused heart producing the effluent and the trabecula on which the effluent was assayed. Therefore, endothelial cells are required for the presence of cardioactive substances in the coronary effluent. The production of a labile endothelium-derived upregulating (positively inotropic) factor and a more stable endothelium-derived downregulating (negatively inotropic) factor has been demonstrated and appears to account for all of the changes in myocardial contractility produced by the coronary effluent. Neither of the endothelium-derived substances demonstrated in the isolated perfused heart is nitric oxide or endothelin. The concentration of the endothelium-derived upregulating factor is sensitive to oxygen tension, whereas the concentration of the endothelium-derived downregulating factor is sensitive to the rate of coronary flow but not oxygen tension. The coronary effluent appears to contain substances that stimulate secretion by the endothelial cells (preendothelial factors) as well as substances that have been produced by the endothelial cells (endothelial factors). The results indicate that during the passage of perfusion medium through the coronary vasculature upregulating and downregulating factors are added to the perfusate in relative concentrations that depend at least in part on local tissue PO2 and the rate of coronary flow. In the intact heart, this mechanism could operate to maintain balance between energy supply and work performed.