This study addresses the question of whether loss of p16INK4 expression contributes to the immortalization of human cells. In vitro immortalization usually proceeds through two phases. In the first phase (lifespan extension), cells continue proliferating and their telomeres continue shortening beyond the point at which normal cells become senescent. In the second phase (immortalization), the cells activate a telomere maintenance mechanism and acquire an unlimited proliferative potential. It has previously been shown that immortalized cells containing viral oncoproteins that bind and inactivate p110RB contain wild-type p16INK4; we therefore examined the p16INK4 status of cell lines that became immortalized in vitro in the absence of these oncoproteins. Three such lines were identified: III-CF/.2A1 and III-CF/E6A2 (both derived from Li-Fraumeni syndrome fibroblasts, probably by spontaneous immortalization) and MePV-231 (normal mesothelial cells transfected with HPV-16 E6/E7 genes that underwent deletion of these genes before immortalization). In each case p16INK4 expression was lost at or before immortalization. Further, a cell strain was identified that had an extended but finite lifespan associated with loss of p16INK4 (and p53) expression. Thus loss of p16INK4 expression was associated with extended in vitro lifespan but was not sufficient for immortalization, even in the absence of wild-type p53.