Renal PEPT1 and PEPT2 cotransporters play an important role in the balance of circulating body oligopeptides and certain peptide like drugs. The reduction in renal functions associated with ageing can affect reabsorption/ excretion balance. Several studies report the importance of adjusting protein intake and optimizing drug dosages for individuals with compromised renal function to avoid adverse reactions. We aim to comprehensively investigate age related changes of PEPT co-transporters at gene, protein and functional level in two important regions of the kidney superficial cortex and outer medulla. A standard method is used to isolate brush border membrane vesicles (BBMV) and outer medulla membrane vesicles (OMMV) from the kidneys of young, middle-aged and old Wistar rats. Different biomolecular techniques are used to determine age-related changes of PEPT cotransporters from different angles: conventional and real-time RT-qPCR are utilized for characterizing the gene expressions; chemiluminescent Western blotting and Immunohistofluorescence are used for relatively quantifying and localizing the protein expressions; fluorescence-based methods were developed to measure the transport activity across BBMV and OMMV. The protein expression of PEPT1 was not only increased in BBMV from old rats, but PEPT1 also appeared in OMMV from middle-aged and old rats. SLC15A1 gene expression in the renal cortex increased in middle-aged group.