Many of the new tool drugs useful for the study of molecular mechanisms of ischemic preconditioning (IP) are very valuable in in vitro systems but produce undesired side-effects after systemic injection in intact animals that limit their applicability. Our aim was to develop an experimental in vivo model that allows the use of said drugs in sufficiently high local concentrations, but avoiding at the same time the systemic side-effects. Several techniques were combined to study regional damage or protection as a result of local drug infusion such as nuclear staining, NADH fluorescence, fluorescent microspheres and tetrazolium salts. In open-chest pigs, the intramyocardial infusion (20 microliters/min) of the adenosine A1-receptor agonist N6-cyclohexyladenosine (0.3 mmol) for 10 min prior to a 60-min LAD-occlusion and 120-min reperfusion mimicked IP by exerting a local protection (n = 9, p < 0.001). Krebs-Henseleit buffer (negative control) was without protective effect. IP's cardioprotection was locally prevented by the intramyocardial application of the adenosine A1-receptor antagonist cyclopentyltheophylline (1 mmol, infused during IP; n = 6, p < 0.001) but not by KHB. The protein kinase C (PKC)-inhibitors staurosporine (100 nmol, n = 6) or bisindolylmaleimide (BIS, 25 mumol, n = 9) did not prevent IP locally. The PKC activator phorbol myristate acetate (PMA, 1 mumol, n = 6) was ineffective in preventing ischemic injury and increased the amount of necrosis in IP, whereas BIS exerted a local myocardial protection (n = 9, p < 0.001). In conclusion, the new model of intramyocardial infusion appears to be useful for the investigation of IP's signal transduction. Our data support the role of the adenosine A1-receptor in IP, but suggest that inhibition instead of activation of PKC may protect ischemic myocardium from infarction.