Adenosine released during cardiac ischemia exerts a potent, protective effect in the heart via activation of A1 or A3 receptors. However, the interaction between the two cardioprotective adenosine receptors and the question of which receptor is the more important anti-ischemic receptor remain largely unexplored. The objective of this study was to test the hypothesis that activation of both receptors exerts a cardioprotective effect that is significantly greater than activation of either receptor individually. This was accomplished by using a novel design in which new binary conjugates of adenosine A1 and A3 receptor agonists were synthesized and tested in a novel cardiac myocyte model of adenosine-elicited cardioprotection. Binary drugs having mixed selectivity for both A1 and A3 receptors were created through the covalent linking of functionalized congeners of adenosine agonists, each being selective for either the A1 or A3 receptor subtype. MRS 1740 and MRS 1741, thiourea-linked, regioisomers of a binary conjugate, were highly potent and selective in radioligand binding assays for A1 and A3 receptors (Ki values of 0.7 ? 3.5 nM) versus A2A receptors. The myocyte models utilized cultured chick embryo cells, either ventricular cells expressing native adenosine A1 and A3 receptors, or engineered atrial cells, in which either human A3 receptors alone or both human A1 and A3 receptors were expressed. The binary agonist MRS1741 can co-activate A1 and A3 receptors simultaneously, with full cardioprotection (EC50 ~0.1 nM) dependent on expression of both receptors. Thus, co-activation of both adenosine A1 and A3 receptors by the binary A1/A3 agonists represents a novel general cardioprotective approach for the treatment of myocardial ischemia.