We have identi®ed a 360 kb YAC that carries a cell senescence gene, SEN16. In our earlier studies, we localized SEN16 within a genetic interval of 3 ± 7 cM at 16q24.3. Six overlapping YACs spanning the chromosomal region of senescence activity, were assembled in a contig. Candidate YACs, identi®ed by the markers located in the vicinity of SEN16, were retro®tted to introduce a neo selectable marker. Retro®tted YACs were ®rst transferred into mouse A9 cells to generate A9/YAC hybrids. YAC DNA present in A9/YAC hybrids was further transferred by microcell fusion into immortal cell lines derived from human and rat mammary tumors. YAC d792t2 restored senescence in both human and rat mammary tumor cell lines, while an unrelated YAC from chromosome 6q had no senescence activity. Oncogene (2000) 19, 217 ± 222.