Four INK4 proteins can prevent cell proliferation by specifically inhibiting cyclin D-dependent kinases. Both p18INK4c and p19INK4d were widely expressed during mouse embryogenesis, but p16INK4a and p15INK4b were not readily detected prenatally. Although p15INK4b, p18INK4c and p19INK4d were demonstrated in many tissues by 4 weeks after birth, p16INK4a 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 p19ARF were not detected before birth but were ubiquitous postnatally. Expression of p16INK4a and p15INK4b was induced when mouse embryos were disrupted and cultured as mouse embryo 'fibroblasts' (MEFs). The levels of p16INK4a and p18INK4c, but not p15INK4b or p19INK4d, 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 p16INK4a. In contrast, one MEF line that sustained bi-allelic deletions of INK4a initially remained diploid. Therefore, loss of p16INK4a and other events predisposing to polyploidy may represent alternative processes contributing to cell immortalization. Whereas p18INK4c and p19INK4d may regulate pre- and postnatal development, p16INK4a more likely plays a checkpoint function during cell senescence that underscores its selective role as a tumor suppressor.