Myocardial ischemia with limited oxygen supply and poor washout of metabolites results in varying levels of cell death (infarct). Although rapid re-establishment of blood flow (reperfuse) is necessary, paradoxally reperfusion per se may lead to further injury to the heart, defined as ischemia/reperfusion injury. During the last few years, we have demonstrated that mitogen insulin can activate cell survival programmes and attenuate apoptosis, a mechanism involving the pro-survival serine/threonine protein kinase B (PKB/Akt). A well known substrate for PKB/Akt is the constitutively active serine/threonine kinase Glycogen synthase kinase-3b (GSK3b). GSK3b negatively regulates downstream signalling mechanisms, therefore phosphorylation and thereby inactivation of GSK3b stimulates many cellular functions by removing the negative constraint imposed by GSK3b. Recently, studies have suggested that phosphorylation of GSK3b enhances myocardial tolerance against ischemia/reperfusion. In this study we wanted to verify this postulation by using a GSK3b inhibitor. In addition we wanted to determine whether GSK3b inhibition affords additional cardioprotection together with Insulin. Ischemia-reperfusion was induced in ex vivo rat hearts using the Langendorff perfusion procedure. Hearts were subjected to 30 min of regional ischemia followed by 120 min of reperfusion with or without the administration of GSK3b inhibitor or Insulin alone or in combination. Infarct size/area at risk was measured using the standard Evans Blue dye (0.2%) and triphenyltetrazolium (TTC) staining. Exposing the hearts to GSK3b inhibitor or Insulin at reperfusion significantly reduced infarct size. Combination treatment was significantly different from control, however it did not have an additive cardioprotective effect compared to the two agents alone.