Telomeres are specialized DNA/protein complexes that cap eukaryotic chromosome ends as T-loop structures and maintain genomic integrity. Vertebrate telomeric DNA consists of tandem double-strand repeats which terminate in a 3 0 single-strand G-rich overhang. The telomeric 3 0-overhang is important for the formation of the T-loop. In mammalian mortal somatic cells, telomeres shorten with each successive division and contribute to the onset of replicative senescence. The exact molecular mechanism underlying replicative senes-cence remains unclear: whether telomere shortening is the only trigger or loss of telomeric 3 0-overhang plays a causal role. To further address this issue, we investigated telomeric 3 0-overhang and telomere changes during cell proliferation toward replicative senescence. We demonstrate here that telomeric 3 0-overhang, similar to telomeres, exhibits progressive attrition with each cell division in primary sheep fibroblasts and that telomeric 3 0-overhang size does not determine the rate of telomere shortening. Furthermore, the sizes of telo-meric 3 0-overhangs are associated with telomere lengths. Our results suggest that alteration of the 3 0-overhang and the telomere during cellular proliferation are associated. Together they may contribute to maintain chromosomal stability and to regulate replicative senescence.