Purpose: Rat neonatal hearts (14 days old) are more resistant to ischaemia and reperfusion (I/R) compared to adult. Generation of reactive oxygen species (ROS) during I/R is a key determinant of injury. Therefore antioxidants, including catalase could underlie this age-related difference in vulnerability to I/R. Consistent with this is our recent finding that cardiac catalase levels are higher in neonatal rats compared to adult. The aim of this work was to determine whether this difference in catalase expression alters the rate of ROS production in both quiescent and stressed cardiomyocytes in the presence or absence of a catalase inhibitor.Methods: Cardiomyocytes were isolated from 14 day old (neonate) and 250 g (adult) male Wistar rats using a collagenase and protease digestion method. Isolated cardiomyocytes were loaded with a ROS detecting fluorescent dye (DCF) and then resuspended in a HEPES buffer containing 2.5 mM CaCl2. Cardiomyocytes were then incubated at 37°C for 30 min with and without the addition of 20 mM 3-amino-1,2,4-triazole (3-AT) (a catalase inhibitor). Cardiomyocytes were aliquoted into a 96-well plate (2000 viable cells per well). The wells were excited with 485 nm and emission detected at 520 nm using a fluorescent plate reader. DCF fluorescence was measured every 2 min for 20 min and the rate of DCF fluorescence was calculated. Some wells had 10 µM H2O2 added immediately prior to the 20 min recording period.Results: At basal levels (without H2O2) the rate of DCF fluorescence (ROS levels) in cardiomyocytes was similar for both age groups. However, upon addition of H2O2 the rate of DCF fluorescence increased significantly more in the adult cardiomyocytes compared to the neonate cardiomyocytes (P < 0.001). Inhibition of catalase by 3-AT in quiescent cardiomyocytes increased the rate of ROS production to a similar degree for both groups. In contrast, addition of H2O2 in the presence of a catalase inhibitor resulted in a significantly larger increase in ROS levels in neonatal cardiomyocytes compared to adult (P < 0.001).Conclusions: During stress with H2O2, ROS production was more marked in adult cardiomyocytes compared to neonatal which is consistent with the higher levels of catalase in neonatal heart. The role of catalase was further confirmed by the finding that catalase inhibition resulted in a larger increase in ROS production in neonatal cardiomyocytes compared to adult. These data suggest that the difference in vulnerability between adult and neonatal hearts could be in part due to different catalase levels and that its role becomes evident during stress.