In whole heart preparations, the sulphydryl containing amino acid, L-cysteine has been shown to increase glutathione levels (Roberts and Francetic, 1991), and aid free radical scavenging during ischaemia reperfusion (Tang et al. 1991). There is little known however about how these cardioprotective actions of L-cysteine in whole hearts are related to events at the cellular level. The main aims of this study were to investigate the effect of incubation with L-cysteine on intracellular glutathione (GSH) levels and on contractile changes during oxidative stress in isolated ventricular myocytes. Male Wistar rats were humanely killed by cervical dislocation and the hearts dissected. Ventricular myocytes were isolated as described previously (King et al. 2003). One group of cells were incubated ± 0.5mM L-cysteine in the presence/absence of 0.1mM diethylmaleate (a thiol modifying agent that indirectly causes oxidant stress, Li et al. 1999) overnight followed by measurement of glutathione using a Calbiochem kit. Cells from a separate isolation were incubated in 0, 0.5, 2.5 or 5mM L-cysteine for 2 hours at 20°C. These cells were then placed in suspension on the stage of a fluorescence microscope at 37°C and stimulated at 0.2Hz (King et al. 2003). They were then exposed to oxidative stress by superfusing with a Tyrode solution containing 0.2mM H₂O₂.Measurements were taken of the time required for a field of myocytes to undergo arrhythmia. The effect of L-cysteine incubation on the intracellular L-cysteine concentration was measured using HPLC. Incubation with L-cysteine significantly increased the intracellular glutathione (GSH) level both in the presence and absence of diethylmaleate (DEM). Intracellular GSH in cells incubated with 0.5mM cysteine was 2.58 ± 0.24 nmol/mg protein, compared to 1.55 ± 0.08 nmol/mg (N = 6 ± S.E., p<0.01, ANOVA) in control cells. In the presence of DEM, GSH levels in cells incubated with 0.5mM L-cysteine was 1.94 ± 0.26 nmol/mg protein compared to 1.12 ± 0.07 nmol/mg protein (N = 6 ± S.E., p<0.05) in control cells without L-cysteine. There was also a significant increase in the time to arrhythmia in cells incubated with different L-cysteine concentrations compared to control cells without L-cysteine. These results suggest that L-cysteine protects isolated cardiomyocytes during oxidative stress by maintaining intracellular glutathione levels.