Normal cells have a finite replicative capacity. Cells capable of proliferation in vivo will initially grow in culture, but their proliferative capacity progressively declines with successive passage in culture, eventually leading to the cessation of growth. This phenomenon of finite life span has been termed replicative senescence. Although senescence is thought to be irreversible, there are conditions in which cells may bypass senesence or extend their replicative lifespan. In contrast to human cells which have never been observed to spontaneously immortalize, rodent cells bypass senescence at a relatively high frequency. The mechanism by which cells undergo replicative senenescence, and the reason why the rodent system is more permissive to immortalization and tumorigenesis is not understood. Recently, we generated mice lacking components of the DNA double-strand break repair apparatus (Ku80-/- and Ku70-/- mice). Ku-deficient mice display a profound defect in cellular proliferation: knockout mice are approximately 50% the size of their littermates, exhibit features that resemble premature aging, and embryonic Ku-/- fibroblasts undergo premature senescence. These observations suggested that the Ku protein may normally play a role in senescence and the transition to the immortal phenotype. To study the relationship between DNA repair, senescence and immortalization, we isolated wildtype mouse embryo fibroblasts (MEFs), passaged them serially in culture, and monitored their replicative capacity. With continued passage, cells entered a slow growth senescent phase (passage 5-7). By passage 9, the cells emerged from crisis, regained their initial growth rate and generated immortalized cultures. We observed that Ku protein levels correlated inversely with growth potential during serial passage. Whereas Ku levels were abundant in early and late passage cultures, Ku decreased to undetectable levels by passage 5 (just prior to senescence). However, cessation of growth did not invariably lead to the down regulation of Ku protein: levels of Ku did not decline when early pre-senescent or late post crisis cells were made quiescent or confluent. As would be predicted from these results, wildtype passage 6 senescent cells were more sensitive to DNA damaging agents than early and late passage cells, and exhibited a similar dose response as Ku-deficient MEFs. In summary, these data suggest that immortalization of MEFs (which has been postulated to be mutational in nature) may be promoted by defective DNA repair during senescence. It remains to be demonstrated that the restoration of normal DNA repair capacity to MEFs throughout passage (by transfection experiments currently in progress) would make MEFs less permissive to immortalization and result in the extension their lifespan up to the point in which the deterioration of telomere length (rather than global DNA damage) would trigger senescence. In this case, the replicative capacity and behavior of mouse cells would resemble their human counterparts.