The uncontrolled generation of reactive oxygen species is a major cause of ischaemia-reperfusion injury (Dhalla et al. 2000). We have previously shown that the sulphydryl containing amino acid cysteine is protective against ischaemia reperfusion in whole heart (Schackebaei et al. 2004) and against oxidative stress in isolated cardiomyocytes (Evans et al. 2004). However, the mechanisms underlying the protective effects of cysteine remain unclear. The aim of this study was to test the hypothesis that cysteine increases activity of glutathione peroxidase (GPx). Male Wistar rats were humanely killed and ventricular myocytes isolated as described previously (King et al. 2003). Cells were incubated with or without (control) 0.5mM L-cysteine for 2 h at room temperature. Myocytes were then placed in suspension on the stage of a microscope at 37°C and stimulated at 0.2Hz. They were exposed to oxidative stress by superfusion with a Tyrode solution containing 0.2mM H₂O₂. Measurements were taken of the time to hypercontracture. In a separate series of experiments GPx was extracted, isolated and quantified as described previously (King et al. 2003). Incubation with cysteine significantly increased time to hypercontracture during oxidative stress (control 885±51.2 s vs. cysteine incubation 1108±0.234 s, p<0.001 (unpaired t test), n=4, mean±SEM). Additionally, the activity of the enzyme GPx was significantly greater in the cysteine incubated cells (1.108±0.234 mU/mg protein) compared to the control cells (0.538±0.1 mU/mg protein, p<0.05 (unpaired t test), n =4-5, mean±SEM). These results provide further evidence that incubation with 0.5mM cysteine protects against oxidative stress in isolated cardiomyocytes and that this may be as a result of stimulated GPx activity.