Cultured primary human cells, which lack telomerase, enter a state of replicative senescence after a characteristic number of population doublings. During this process telomeres shorten to a critical length of approximately 5-7 kb. The mechanistic relationship between advanced cell passage, cellular senescence and telomeric function has yet to be fully elucidated. In the study described here, we investigated the relationship between changes in telomeric replication timing and/or sister chromatid separation at telomeric regions and advanced cell passage. Using fluorescence in situ hybridization, we analyzed the appearance of double hybridization signals (doublets), which indicate that the region of interest has replicated and the replicated products have separated sufficiently to be resolved as two distinct signals. The results showed that the replication and separation of several telomeric regions occurs during the second half of S-phase and that a delay in replication and/or separation of sister chromatids at these regions occurs in pre-senescent human fibroblasts. Surprisingly, in a significant percentage of pre-senescent cells, several telomeric regions did not hybridize as doublets even in metaphase chromosomes. This delay was not associated with extensive changes in methylation levels at subtelomeric regions and was circumvented in human fibroblasts expressing ectopic telomerase. We propose that incomplete replication and/or separation of telomeric regions in metaphase may be associated with proliferative arrest of senescent cells. This cell growth arrest may result from the activation of a mitotic checkpoint, or from chromosomal instability consequent to progression in the cell cycle despite failure to replicate and/or separate these regions completely.