The renal H+/peptide cotransporters (PEPT1 and PEPT2) play important roles in the reabsorption of di- and tri- peptides. Based os structural similarity, PEPT1 and PEPT2 are also capable of transporting a broad range of peptidomimetic drugs including certain angiotensin converting enzyme (ACE) inhibitors, ?-lactam antibiotics, anti-tumor and anti-viral agents. In the present study, the effect of ageing on L-glycyl-L-glutamine (gly-gln) transport was measured using a novel approach involving the use of the fluorescent indicator, 3,3-Dipropylthiacarbocyanine iodide (DiS-C3-(3)) in membrane vesicles isolated from the superficial cortex and outer medulla of two groups of male Wister rats of approximately 6-weeks (young) and 9-months (middle-aged) old. This study showed for the first time that DiS-C3-(3) fluorescence could be used as a reporter of the membrane potential difference in membrane vesicles isolated from different kidney regions and from rats of different ages. Furthermore it was shown that gly-gln uptake was consistently greater in the younger age group and in the superficial cortex compared to the outer medulla.