PURPOSE. To demonstrate a relationship between telomere lengths and levels of p53 in cultured bovine corneal endothelial cells (CECs) during aging. METHODS. Bovine CECs were grown and aged as long-term cultures. Telomere lengths were determined directly on gels with 32P probes after treatment of isolated DNA with RsaI and HinfI. Protein p53 was determined using an enzyme-linked immunosorbent sandwich assay. Cellular aging and the development of replicative senescence were monitored by the appearance of senescent morphology and the β-galactosidase assay. RESULTS. Bovine CEC telomeres lost 4 kb (from 12.8 to 8.8 kb) over 1 year (89 population doublings [PDs]). The p53 levels in bovine CECs were initially small (~60 pg/million cells), but rose 3.5-fold by culture age of 260 days (64 PDs). On initiation, cultured bovine CECs did not stain for the senescent marker β- galactosidase. However, these cells stained at 89 PDs and senescent morphology was observed in the cultures at 64 PDs. CONCLUSIONS. The data indicate an inverse relationship between telomere lengths (decreasing) and levels of p53 (increasing) in bovine CECs during aging. These properties may influence the ability of these cells to divide as they enter into replicative senescence.