Some dipeptides have been implicated in myocardial protection, but little is known about their membrane transporter PEPT2. The aim of this study was to determine whether the expression and activity of the cardiac-type PEPT2 cotransporter could be affected by ageing and/or hypertension. Sarcolemmal vesicles (SV) were isolated from the hearts of all rat groups using a standard procedure to investigate the transport activity and protein abundance by fluorescence spectroscopy and Western blot, respectively. SLC15A2 "PEPT2" gene expression was relatively quantified by RT-qPCR. In the Wistar rat groups, the protein and gene expression of PEPT2 were upregulated with ageing. These changes were accompanied by corresponding increases in the competitive inhibition and the transport rate (Vmax) of β-Ala-Lys (AMCA) into SV isolated from middle-aged hearts. Although, the transport rate of β-Ala-Lys (AMCA) into SV isolated from old hearts was significantly the lowest compared to middle-aged and young adult hearts, the inhibition percentage of β-Ala-Lys (AMCA) transport by Gly-Gln was the highest. In the WKY and SHR rat groups, Y-SHR hypertrophied hearts showed an increase in PEPT2 gene expression accompanied by a significant decrease in protein expression and activity. With advanced age, however, M-SHR hypertrophied hearts revealed significantly lower gene expression, but higher protein expression and activity than Y-SHR hearts. These findings suggest that increased expression of PEPT2 cotransporter in all types of middle-aged hearts could be exploited to facilitate di-and tripeptide transport by PEPT2 in these hearts, which subsequently could result in improved myocardial protection in these populations.