It has been proposed that the progressive shortening of telomeres in somatic cells eventually results in senescence. Previous experiments have demonstrated that many immortal cell lines have acquired telomerase activity leading to stabilization of telomere length. Telomere dynamics and telomerase activity were examined in the telomerase-positive immortal cell lines HeLa and 293 and subclones derived from them. A mass culture of HeLa cells had a stable mean telomere length over 60 population doublings (PD) in vitro. Subclones of this culture, however, had a range of mean telomere lengths indicating that telomeric heterogeneity exists within a population with a stable mean telomere length. Some of the subclones lacked detectable telomerase activity soon after isolation but regained it by PD 18, suggesting that at least some of the variation in telomere length can be attributed to variations in telomerase activity levels. 293 subclones also varied in telomere length and telomerase activity. Some telomerase-positive 293 subclones contained long telomeres that gradually shortened, demonstrating that factors other than telomerase also act to modulate telomere length. Fluctuations in telomere length in telomerase-positive immortalized cells may contribute to chromosomal instability and clonal evolution.