The non-essential amino acid, L-cysteine has been implicated in myocardial protection both as an antioxidant and as an important regulator of the intracellular glutathione pool (Tang et al. 1991). Little however is known about the molecular and functional characteristics of L-cysteine transport in heart. The aim of this study was to use isolated rat cardiomyocytes to investigate the expression of a candidate transporter, ASCT2 and to investigate L-cysteine uptake.Male wistar rats were humanely killed by cervical dislocation. Ventricular cardiomyocytes were isolated by enzyme digestion (King et al. 2001). Total RNA was isolated from cardiomyocytes using TRI-reagent, followed by cDNA synthesis using the Retroscript Kit (Ambio) and reverse transcription polymerase chain reaction (RT PCR), which was carried out using primers directed against ASCT2. Separate suspensions of cardiomyocytes were used to measure the uptake of L-[³⁵S]cysteine by oil filtration centrifuge stop technique (King et al. 2001). Homogenisation and differential centrifugation were used to prepare cardiac sarcolemmal vesicles (King et al. 2001) and brain synaptosomal membrane vesicles (Kanner, 1978), which were then used in Western blotting. The anti-ASCT2 antibody used for this was produced by the same procedure as described previously except that the sequence of the injected peptide corresponded to the C terminal portion of ASCT2 (Nicholson & McGivan, 1996). Following gel electrophoresis of RT PCR products, a single band of the expected molecular weight was present in the cardiac myocytes. The anti-ASCT2 antibody recognised a protein of appropriate size (56 KDa) in both cardiac sarcolemmal vesicles and brain synaptosomal vesicles (used as a positive control). At 0.5min the uptake of 0.1mM L-[³⁵S]cysteine in sodium containing media was 467.4 ± 35.9 pmol/mg protein, which was significantly greater than the 187 ± 16.4 pmol/mg protein (p < 0.001, n = 5 ± S.E. Students ttest) measured in sodium free media (sodium replaced isosmotically with choline). The Km and Vmax of the sodium dependent component of cysteine uptake were 179.5 ± 3.0mM and 1399.9 ± 50.8 pmol/min/mg protein (n = 4 ± S.E.) respectively. These results suggest that the amino acid transporter, ASCT2 is expressed in different heart preparations, and that cysteine uptake into rat cardiomyocytes is strongly sodium dependent.