Telomeres, which are specialized structures consisting of T2AG3 repeats and proteins at the ends of chromosomes, may be essential for genomic stability. To test whether telomere length maintenance preserves genomic stability in rats (Rattus rattus and Fischer 344), we assayed telomerase activity and telomere length in the rat hepatic epithelial stem-like cell line WB-F344 during aging in vitro and in tumor-derived lines. Telomerase activity in the parental WB-F344 line was repressed at low and intermediate passage levels in vitro and reexpressed at high passages. Southern blot hybridization and quantitative fluorescence in situ hybridization analyses demonstrated that telomeres were significantly eroded at intermediate passage levels, when telomerase was repressed, and at high passage levels, when telomerase was expressed. Fluorescence in situ hybridization analysis also revealed interstitial telomeric sequences in rat chromosomes. Tumor-derived WB-F344 cell lines that express telomerase had variably shortened telomeres. Cytogenetic analyses performed on WB-F344 cells at low, intermediate, and high passages demonstrated that chromosome instability was most severe in the intermediate passage cells. These data suggest that telomere shortening during aging of rat hepatic epithelial stem-like WB-F344 cells in vitro and during selection of tumorigenic lines in vivo may destabilize chromosomes. Expression of telomerase in high passage cells appeared to partially stabilize chromosomes.