To investigate the basic mechanism for formation of adenosine, cardiac work was increased using an isolated guinea pig working heart preparation. Cardiac metabolism was stimulated by intracoronary infusion of isoproterenol, norepinephrine (NE), ouabain, and the cardiotonic agent 1H-imidazo[4,5-b]pyridine, 2-[2-methoxy-4-(methylsulfinyl)-phenyl] (AR-L 115), and the release of adenosine into the effluent was determined. Besides their inotropic effects on myocardial tissue, these substances affect differently the tone of coronary arteries. Thus they influence the supply-to-demand ratio for O2 and the tissue cyclic AMP content differently. When myocardial O2 consumption was increased to the same extent, the changes in coronary flow induced followed the order of AR-L 115 greater than isoproterenol greater than NE greater than ouabain. Conversely, the rank order of potency causing adenosine and inosine release was NE greater than ouabain greater than isoproterenol greater than AR-L 115. When NE-induced vasoconstriction was abolished by prazosin, the release of adenosine and inosine was significantly diminished. Enhancement of O2 supply by overperfusion of the coronary arteries in isoproterenol-stimulated hearts was associated with a significant reduction of nucleoside release into the effluent. Our findings suggest that the major stimulus for myocardial adenosine formation is an imbalance between O2 delivery and O2 demand and not the metabolic rate as such. In the isolated heart, adenosine is formed as a feedback signal and can compete with NE- and ouabain-induced vasoconstriction.