Amino acids are used in the heart for protein synthesis, energy production and signal transduction (1). This is facilitated by amino acid transport across the cardiac sarcolemma. In the kidney and small intestine, the proton coupled transporters, PEPT1 (gene name SLC15A1) and PEPT2 (gene name SLC15A2) provide an extremely efficient mechanism for the transport of bulk quantities of amino acids in the form of small peptides (2). PEPT1 and PEPT2 are also important pharmacologically since they transport peptidomimetic compounds such as angiotensin converting enzyme (ACE) inhibitors and β-lactam antibiotics (2). The aim of this investigation was to investigate whether these transporters are expressed and active in isolated ventricular cardiomyocytes. Ventricular cardiomyocytes were isolated from adult male guinea-pigs by enzyme and mechanical dispersion techniques as described previously (3). The oil filtration centrifuge stop technique used to measure the uptake of 0-3mM L-glycyl-L-[¹⁴C]sarcosine (gly-sar) into the cardiomyocytes has also been described (4). The only modification to the published method (4) was the use of Mes/Tris instead of Hepes/Tris to prepare solutions with pH ≤ 6.5. Reverse transcription polymerase chain reaction (5) was used to investigate the expression of PEPT1 and PEPT2 in the cardiomyocytes. Results are expressed in terms of intracellular space (i.e. as pmol/μl intracellular space as described in (4)) and are means ± S.E.M. of 4 or 5 experiments. The uptake of 200μM gly-sar at 30 s after dipeptide addition was strongly dependent on external pH with transport greatest at pH 5.75 (217.8 ± 15.8 pmol/μl), and least at pH 8.0 (72.1 ± 13.4 pmol/μl, p<0.01, t test). At pH 6.0, the initial rate of gly-sar uptake obeyed Michaelis-Menten kinetics with a K_m of 495.5 ± 69.6μM and a V_max of 1470.5 ± 69.6 pmol/μl/min. The control initial rate of gly-sar uptake at pH 6.0 of 343.8 ± 7.5 pmol/μl/min, was significantly reduced by the addition of 10mM of the ACE inhibitor, enalapril (92.2 ± 31.2 pmol/μl/min); or of the β-lactam antibiotics, cefadroxil (82.9 ± 32.7 pmol/μl/min), cephaloridine (116.7 ± 11.6 pmol/μl/min) or ampicillin (103.5 ± 12.2 pmol/μl/min); or of various L-amino acid containing di- or tri-peptides (all comparisons, p<0.01, ANOVA with a Dunnett post test). In contrast, 10mM of the constituent amino acids, glycine (336.2 ± 11.1 pmol/μl/min) or sarcosine (339.4 ± 18.2 pmol/μl/min) or of the D-amino acid containing dipeptide D-ala-D-ala (367.2 ± 25 pmol/μl/min) did not significantly alter the control uptake. PEPT2 specific primers recognised mRNA of the appropriate size in kidney (positive control) and cardiomyocytes, whilst PEPT1 was expressed in kidney but not cardiomyocytes. These results provide the first evidence supporting the presence of a functional dipeptide transporter in isolated cardiomyocytes.