NO is an important bioactive molecule involved in a variety of physio- and pathological processes, including apoptosis induction. The proapoptotic activity of NO involves the rise in the tumor suppressor p53 and the accumulation and targeting of proapoptotic members of the Bcl-2 family, in particular Bax and the release of cytochrome c from the mitochondria. However, the exact mechanism by which NO induces p53 activation has not been fully elucidated. In this study, we describe that NO induces p19(ARF) through a transcriptional mechanism. This up-regulation of p19(ARF) activates p53, leading to apoptosis. The importance of p19(ARF) on NO-dependent apoptosis was revealed by the finding that various cell types from alternate reading frame-knockout mice exhibit a diminished response to NO-mediated apoptosis when compared with normal mice. Moreover, the biological relevance of alternative reading frame to p53 apoptosis was confirmed in in vivo models of apoptosis. Together, these results demonstrate that NO-dependent apoptosis requires, in part, the activation of p19(ARF).