Abstract We have investigated whether the average relative telomere length of lens epithelial cells (LECs) from brown Norway rats decreases with the age of the donor animal, and whether chronic caloric restriction (CR) of the rats delays the telomere shortening. Our previous studies have demonstrated that clonal proliferative potential of rodent LECs as well as the in vivo rate of DNA synthesis decreases with age and that this decrease is slowed by chronic lifelong caloric restriction (CR). In order to determine if telomeric shortening might be involved in this loss of proliferative potential, we examined relative telomeric lengths in young, old ad lib fed (AL), and old calorically restricted (CR) brown Norway rats. We used fluorescence in situ hybridization with a peptide nucleic acid probe (PNA) complementary to the telomeric repeat sequence to quantitate relative telomere lengths in LECs in lens sections (TELO-FISH). Control experiments demonstrated that the PNA probe binding was restricted almost entirely to the terminal portions of the rat chromosomes with less than 5% bound at interstitial sites in typical metaphase spreads. The relative telomere lengths of interphase human fibroblast standards, as determined by TELO-FISH, were in good agreement with terminal restriction fragment analyses of the same standards and with literature values for rat cells. The average telomere lengths of interphase nuclei in the old AL rat LECs were found to be 21% shorter than paired young AL controls ( P < 0.01 by Wilcoxian signed rank test). The calorically restricted old rats had less telomere erosion (12%) than the old AL group ( P < 0.05). Although it is not clear whether such moderate telomeric erosion can limit cell division in rodent LECs, the telomeric shortening correlated well with previous studies demonstrating reduced clonal, replicative potential, and reduced rates of in vivo DNA replication in LECs from old rodents and a delay in this attenuation in animals on chronic CR.