This study describes the first example of spontaneously immortalized, telomerase-negative hamster cells which replicated in the absence of telomere shortening. Primary hamster buccal pouch epithelial (HBPE) cells were subcultured for approximately 10 population doublings (PDs) before they ceased to divide. Among the non-replicating HBPE cells, a colony of proliferating cells had appeared. These cells named HBPE-SI (spontaneously immortalized) replicated with a mean doubling time of 27 h for 120 PDs in culture and are still replicating. Surprisingly, telomerase activity and hamTERT (hamster telomerase gene) expression were not detectable in HBPE-SI, although an exogenous hamTERT promoter showed promoter activity in these cells. HBPE-SI cells replicated in the apparent absence of apparent telomere shortening during their entire in vitro culture, indicating telomerase-independent mode of telomere maintenance. Analysis of the hamTERT promoter in HBPE-SI cells revealed hypermethylation. Exposure of the cells to 5-aza-2'-deoxycytidine (5-aza CdR) restored expression of endogenous hamTERT mRNA. These results indicated that HBPE-SI cells maintained their telomere length in the absence of telomerase activity, and that hamTERT gene expression in these cells was silenced by hypermethylation of the promoter.