G protein-coupled receptors for adenosine (A₁, A₃, A_2A, and A_2B), bradykinin (B₁) and opioids (δ) are all involved in the mechanism of ischemic preconditioning. Although the heart is comprised of many tissue types, it has been assumed that preconditioning’s protective signaling occurs in the cardiomyocyte. We critically tested that hypothesis by testing for the presence of each of these receptors in isolated adult rabbit ventricular myocytes that had been transfected with cyclic nucleotide-gated (CNG) ion channels. Because subsarcolemmal cyclic adenosine monophosphate (cAMP) opens the CNG channels, we could monitor cAMP levels within a single cardiomyocyte by measuring channel current with a patch pipette. The presence of a receptor would be confirmed if we could alter cAMP in the cell with a selective agonist to the receptor being studied. Superfusion with the β-adrenergic G_s-coupled receptor agonist isoproterenol (50 nmol/L) transiently increased cAMP levels and, therefore, channel current. Pretreatment with selective agonists to A₁ or A₃ adenosine receptors (ARs) that are G_i-coupled markedly attenuated the response to isoproterenol, indicating inhibition of adenylyl cyclase by increased G_i activity. Agonists to bradykinin or δ-opioid receptors also attenuated isoproterenol’s response. A_2AAR and A_2BAR are G_s-coupled. The A_2AAR–selective agonist CGS21680 increased current through CNG channels but only in the presence of phosphodiesterase (PDE) inhibitors, indicating low surface receptor activity and high intracellular PDE activity. As we previously reported, BAY 60-6583, an A_2BAR-selective agonist which mimics preconditioning’s protection in rabbit heart, neither increased nor decreased membrane current in transfected cardiomyocytes, suggesting the absence or a markedly limited number of A_2BAR in the sarcolemma. However, reverse transcription polymerase chain reaction (RT-PCR) of purified cardiomyocytes yielded an A_2BAR band, implying that rabbit cardiomyocytes do indeed express A_2BAR. These data reveal that all receptors reported to be involved in ischemic preconditioning do exist on or within the cardiomyocyte.