Objective: Myocardial ischemia/reperfusion (I/R) including cardioplegic arrest (CA) has been associated with cardiac apoptosis induction. However, the time course of apoptosis activation and the trigger mechanisms are still unclear. As apoptosis inhibition may represent a novel therapeutic strategy for long-term myocardial preservation, we sought to investigate the time course of apoptosis signal-pathway induction during CA. Methods: Sprague Dawley rats (300-350g) were anesthetised, intubated and mechanically ventilated. CA was initiated by infusion of ice-cold crystalloid solution (Custodiol®, 10 ml/kg) into the aortic root, hearts were rapidly excised and stored for 0, 30, 60 and 120 min in 0.9% sodium chloride solution (28°C). In controls no CA was initiated prior to removal and storage at 28°C. In another group calcium-rich cardioplegia was used and an additional group received a caspase-8 inhibitor prior to CA induction. LV cytosolic extracts were isolated and investigated for the activity of caspase-3 and -6 (effector caspases), and caspase-8 and -9 (involved in extrinsic and intrinsic pathways of apoptosis induction). Fluorometric activity assays were performed using specific substrates. Results: Activities of all tested caspases were significantly increased immediately after CA induction compared to controls. Administration of the caspase-8 inhibitor significantly reduced activities of all caspases. With calcium-rich cardioplegia caspase activities were significantly lower compared to low-calcium CA. Control hearts also showed increase of caspase activities during cold storage ischemia without CA, but had significantly different time courses compared to hearts with CA. Conclusions: Our data show rapid apoptosis signal-pathway induction immediately following CA exposure. Thus, apoptosis signal-pathway inhibition as a potential strategy for improved myocardial preservation would have the greatest effect when applied prior to CA exposure.