Telomeres are nucleoprotein complexes located at chromosome ends, vital for preserving chromosomal integrity. Telomeric DNA shortens with progressive rounds of cell division, culminating in replicative senescence. Previously we have reported, on the basis of fluorescent in situ hybridization, that several human telomeric regions display solitary signals (singlets) in metaphase cells of presenescent fibroblasts, in comparison to other genomic regions that hybridize as twin signals (doublets). In the current study, we show that an additional 12 out of 12 telomeric regions examined also display metaphase singlet signals in pre-senescent cells, and that excess telomere-metaphase singlets also occur in earlier passage cells harvested from elderly individuals. In cancer cell lines expressing telomerase and in pre-senescent fibroblasts ectopically expressing hTERT, this phenomenon is abrogated. Confocal microscope image analysis showed that the telomere metaphase singlets represent regions that have replicated but not separated; this is presumably because of persistent cohesion. The introduction of mutations that interfere with the normal dissolution of cohesion at the metaphase to anaphase transition induced the cut (chromosomes untimely torn) phenotype in early passage fibroblasts, with predominantly telomeric rather than centromeric DNA, present on the chromatin bridges between the daughter nuclei. These results suggest that telomeric regions in animal cells may potentially be sites of persistent cohesion, and that this cohesion may be the basis for an observed excess of fluorescent in situ hybridization metaphase singlets at telomeres. Persistent cohesion at telomeres may be associated with attempted DNA repair or chromosomal abnormalities, which have been described in pre-senescent cells.