Adenosine released during cardiac ischemia exerts a marked protective effect in the heart that is mediated by the A(1) and A(3) subtypes of adenosine receptors. The signaling pathways activated by these adenosine receptors have now been characterized in a chick embryo ventricular myocyte culture model of cardioprotection against ischemia. Selective A(1) and A(3) receptor agonists were shown to activate phospholipases C and D, respectively, to achieve their distinct cardioprotective effects. The specificity of the A(3) receptor-phospholipase D interaction was also demonstrated in chick embryo atrial myocytes (which do not express endogenous A(3) receptors) that had been transfected with a vector encoding the human A(3) receptor. Activation of both endogenous A(1) and A(3) receptors in ventricular myocytes resulted in a protective response greater than that induced by stimulation of either receptor alone. Agonists that activate both adenosine A(1) and A(3) receptors may thus prove beneficial for the treatment of myocardial ischemia.