This experimental study was designed to determine if caspase-3-like protease is activated during a short period of ischemia - reperfusion (I-R) that did not induce apoptosis, and whether protease-3-protease inhibitor could prevent myocardial I-R injury, especially necrotic cell death. The subjects were 20 isolated rat hearts; 10 were pretreated for 20 min with 100 micromol/L of the protease-3-protease inhibitor, peptide antagonist Asp-Glu-Val-Asp-CHO (DEVD) (Group D), and compared with the 10 no-pretreated hearts (Group C). The hearts were then subjected to 20, 30, 45, and 60 min of normothermic global ischemia followed by 30 min of reperfusion. Caspase-3-like protease was significantly elevated after 45 min and 60 min in ischemic hearts. Group D had reduced levels of caspase-3-like protease activity after 45 min and 60 min (302+/-58%, 378+/-69% of pre-ischemic control, respectively), as compared with Group C (542+/-74%, 689+/-85%, respectively) (p<0.05, p<0.05, respectively). Histological analysis also demonstrated a decrease in cellular damage in Group D, as the count ratio of necrotic cells with total cardiomyocytes was 38%, as compared with 78% in the control group (p<0.05). Caspase-3-like protease participated in I-R injury in rat hearts and inhibition of this protease resulted in a reduction of necrotic cell death.