Generation of reactive oxygen species (ROS) during ischaemia and reperfusion (I/R) is a key determinant of cardiomyocytes death. Therefore, anti-oxidants can play a critical role in cardioprotection. The endogenous anti-oxidant enzyme catalase has been implicated in protection but there is no direct evidence showing its involvement. We have recently shown that direct inhibition of endogenous catalase, using 3-Amino-1,2,4-triazole (3-AT), significantly increases the rate of ROS production in freshly isolated ventricular cardiomyocytes (Boles et al., 2014). The aim of the present study was to investigate the effect of catalase inhibition using 3-AT on I/R injury in whole hearts. Experiments were carried out on Langendorff-perfused rat heart. Rats were dispatched by cervical dislocation and hearts immediately extracted. Hearts were subjected to 25 mins stabilization followed by 25 mins global normothermic ischemia (37⁰C) and 120 mins reperfusion. Hearts were assigned to two groups; control (n=4) or treatment with 3-AT (n=4). After 10 mins stabilization, the treatment group was perfused with 15mM 3-AT in Krebs-Henseleit buffer for 15 mins. Perfusate was collected immediately pre-ischemia and at intervals throughout reperfusion in order to determine creatine kinase (CK) activity. Additionally, hearts were stained with Triphenyl tetrazolium chloride at the end of reperfusion to estimate the percentage infarct size. Statistical analysis was performed using a mixed model ANOVA (for CK analysis) and two-tailed unpaired t-test (for infarct percentage). Values are expressed ±SEM. The results showed that catalase inhibition with 3-AT significantly increased both markers of myocardial damage. The mean CK level across all perfusate collections was significantly greater in 3-AT treated hearts than in control hearts (P<0.01). Mean percentage infarct size was significantly greater in 3-AT treated hearts than in control hearts (36.7 ± 4.2 in vs. 17.8 ± 5.4 respectively; P<0.05). These results provide evidence that the catalase inhibitor 3-AT augments I/R injury. This is therefore suggestive of a role for catalase in cardioprotection via the attenuation of ROS accumulation.