The tumor suppressor p16INK4a is a potent mediator of cell cycle arrest in transient expression studies, is induced in senescing cells, and can impose morphological features of senescence. Nonetheless, it is unclear whether p16INK4a can block cell proliferation irreversibly. We explored this issue using osteogenic sarcoma cell clones with inducible p16INK4a expression. Induction of p16INK4a for 1 day arrested most cells in G1 phase. If the induction was then interrupted, p16INK4a levels returned to baseline and robust growth resumed within 3-5 days. When p16INK4a was induced for 6 days DNA synthesis remained strongly inhibited and the cells acquired morphological features of senescence. Moreover, if p16INK4a induction was interrupted at this point and the cells were followed for 12 more days, most cells retained these morphologic features and either failed to divide or died. This occurred despite the prompt return of p16INK4a expression and retinoblastoma protein phosphorylation toward baseline levels. In fact, some senescing cells appeared to enter S phase. These results demonstrate that a sustained period of p16INK4a expression is sufficient in this setting to impose a durable block to cell proliferation and that this state becomes independent of p16INK4a expression, hypophosphorylation of pRB, or a strict G1 arrest.