Controversy exists as to whether platelet activating factor (PAF), a potent phospholipid mediator of inflammation, can actually protect the heart from post-ischemic injury. To determine whether endogenous activation of the PAF receptor is cardioprotective we examined post-ischemic functional recovery in isolated hearts from wild type and PAF receptor knock-out mice. Post-ischemic function was reduced both in hearts with targeted deletion of the PAF receptor and in wild type hearts treated with a PAF receptor antagonist. Furthermore, perfusion with picomolar concentrations of PAF improved postischemic function in hearts from wild type mice. To elucidate the mechanism of a PAF-mediated cardioprotective effect we employed a model of intracellular Ca²⁺ overload and loss of function in non-ischemic ventricular myocytes. We found that PAF receptor activation attenuates both the time-dependent loss of shortening and increases in intracellular Ca²⁺ transients in Ca²⁺ overloaded myocytes. These protective effects of PAF depend on NO, but not activation of cyclic guanosine monophosphate. In addition, we found reversible S-nitrosylation of myocardial proteins must occur in order for PAF to moderate Ca²⁺ overload and loss of myocyte function. Thus, our data are consistent with the hypothesis that low level PAF receptor activation initiates NO-induced S-nitrosylation of Ca²⁺ handling proteins, for example L-type Ca²⁺ channels, to attenuate Ca²⁺ overload during ischemic-reperfusion in the heart. Since inhibition of the PAF protective pathway reduces myocardial postischemic function, our results raise concern that clinical therapies for inflammatory diseases that lead to complete blockade of the PAF receptor may eliminate a significant, endogenous cardioprotective pathway.