Expression of the catalytic subunit of human telomerase (hTERT), in normal human fibroblasts allows them to escape replicative senescence. However, we have observed that populations of hTERT-immortalized human fibroblasts contain 3-20% cells with a senescent morphology. To determine what causes the appearance of these senescent-like cells, we used flow cytometry to select them from the population and analyzed them for various senescence markers, telomere length, and telomerase activity. This subpopulation of cells had elevated levels of p21 and hypophosphorylated Rb, but telomere length was similar to that of the immortal cells in the culture that was sorted. Surprisingly, telomerase activity in the senescent-like cells was significantly elevated compared with immortal cells from the same population, suggesting that high telomerase activity may induce the senescent phenotype. Furthermore, transfection of normal fibroblasts with a hTERT-expressing plasmid that confers high telomerase activity led to the induction of p21, a higher percentage of SA-beta-galactosidase-positive cells, and a greater number of cells entering growth arrest compared with controls. These results suggest that excessive telomerase activity may act as a hyperproliferative signal in cells and induce a senescent phenotype in a manner similar to that seen following overexpression of oncogenic Ras, Raf, and E2F1. Thus, there must be a critical threshold of telomerase activity that permits cell proliferation.