Telomeres, the G/C-rich DNA sequences capping the ends of all eukaryotic chromosomes, have been shown to shorten during replicative aging of normal cells in vitro and in vivo. Moreover, variation in the initial length of terminal restriction fragments (TRF) accounts for much of the variation in replicative capacity of fibroblast cultures from different donors. Since replicative capacity also varies significantly between clones in a mass culture of fibroblasts from a single donor, we wished to further test the hypothesis that the shortening of telomeres to a critical or threshold length acts as a signal for cell senescence. Thus, we measured TRF length and total telomeric signal intensity for 35 clonal fibroblast populations at early passage and at senescence. Replicative capacity was found to be directly proportional to mean TRF length (m = 7.2 population doublings/kbp, r = 0.65, P = 0.0004) and total signal intensity (m = 25.0 population doublings/unit, r = 0.63, P < 0.003) at early passage. More importantly, the variability in both mean TRF length and signal intensity (F = 2.0 and 2.9; P = 0.02 and 0.03, respectively) at senescence was markedly less than that at early passage. Although initial telomere length cannot account for all of the interclonal variability in replicative capacity, our observations support the existence of a critical telomere length in senescing cells and a causal role of telomere shortening in cell senescence.