Four INK4 proteins can prevent cell proliferation by specifically inhibiting cyclin D-dependent kinases. Both p18 INK4c and p19 INK4d were widely expressed during mouse embryogenesis, but p16 INK4a and p15 INK4b were not readily detected prenatally. Although p15 INK4b, p18 INK4c and p19 INK4d were demonstrated in many tissues by 4 weeks after birth, p16 INK4a protein expression was restricted to the lung and spleen of older mice, with increased, more widespread mRNA expression during aging. Transcripts encoding the INK4a alternative reading frame product p19 ARF were not detected before birth but were ubiquitous postnatally. Expression of p16 INK4a and p15 INK4b was induced when mouse embryos were disrupted and cultured as mouse embryofibroblasts'(MEFs). The levels of p16 INK4a and p18 INK4c, but not p15 INK4b or p19 INK4d, further increased as MEFs approached senescence. Following crisis and establishment, three of four independently-derived cell lines became polyploid and expressed higher levels of functional p16 INK4a. In contrast, one MEF line that sustained bi-allelic deletions of INK4a initially remained diploid. Therefore, loss of p16 INK4a and other events predisposing to polyploidy may represent alternative processes contributing to cell immortalization. Whereas p18 INK4c and p19 INK4d may regulate pre-and postnatal development, p16 INK4a more likely plays a checkpoint function during cell senescence that underscores its selective role as a tumor suppressor.