Exogenous expression of the catalytic subunit of telomerase, hTERT, in a normal human foreskin fibroblast cell strain resulted in telomerase activity and an extended proliferative lifespan prior to a period of crisis. Three immortalized cell lines with stably maintained telomere lengths were established from cells that escaped crisis. Each of these cultures underwent a significant downregulation of p16(INK4a) expression due to gene deletion events. One cell line also acquired mutations in both alleles of the p53 tumor suppressor gene. Downregulation of p16(INK4a) and loss of wild-type p53 expression occurred after escape from crisis, so these mutations are most likely not required for immortalization of these cells but rather were selected for during continuous growth in vitro. These findings emphasize the need for caution in the use of hTERT-immortalized cells in studies of normal cell biology or in tissue engineering and the need to monitor for genetic instability and the accumulation of mutations in both the p16(INK4a)/pRb and p53 pathways.