A potential role of p53 in apoptotic signaling is explored, revealing that p53 localizes to mitochondria during p53-dependent apoptosis but not during p53-independent apoptosis or cell cycle arrest. This mitochondrial localization occurs rapidly, within one hour of p53 activation, and precedes changes in mitochondrial membrane potential, cytochrome c release, and procaspase-3 activation. Immunoelectron microscopy and immunofluorescence-activated cell sorter analysis show that most mitochondrial p53 is in the membranous compartment, while a fraction is complexed with the mitochondrial import motor mt hsp70. Induction of ectopic p53 in p53-deficient cells leads to mitochondrial localization, preceding apoptosis. Overexpression of anti-apoptotic Bcl-2 or Bcl-xL blocks mitochondrial p53 accumulation and apoptosis but not cell cycle arrest, suggesting a feedback loop between p53 and mitochondrial apoptotic regulators. Targeting p53 to mitochondria using import leader fusions induces apoptosis in p53-deficient cells, indicating a direct signaling role of p53 at mitochondria. This model proposes that p53 can contribute to apoptosis by direct signaling at mitochondria, amplifying the transcription-dependent apoptosis of p53. The study highlights the importance of mitochondria in p53-mediated apoptosis and suggests a novel role for p53 in direct apoptotic signaling at mitochondria, which are a strategic point in the cell death cascade. The findings support a novel role of p53 in direct apoptotic signaling at mitochondria, enhancing its apoptotic potency. The study also discusses the functional significance of mitochondrial p53 accumulation in p53-mediated apoptosis, showing that mitochondrial p53 localization is specific to p53-dependent apoptosis and not during p53-independent apoptosis or cell cycle arrest. The data suggest a regulatory feedback signaling loop between p53 and Bcl-2 family members at the mitochondrial level. Targeting p53 to mitochondria is sufficient to induce apoptosis in p53-deficient cells, indicating that p53 can launch an apoptotic pathway from the mitochondrial level. The study concludes that p53 has a direct role in a novel apoptosis signaling pathway at the mitochondrion, acting in synergy with the transcription-dependent mode of action of p53, thereby amplifying its apoptotic potency.A potential role of p53 in apoptotic signaling is explored, revealing that p53 localizes to mitochondria during p53-dependent apoptosis but not during p53-independent apoptosis or cell cycle arrest. This mitochondrial localization occurs rapidly, within one hour of p53 activation, and precedes changes in mitochondrial membrane potential, cytochrome c release, and procaspase-3 activation. Immunoelectron microscopy and immunofluorescence-activated cell sorter analysis show that most mitochondrial p53 is in the membranous compartment, while a fraction is complexed with the mitochondrial import motor mt hsp70. Induction of ectopic p53 in p53-deficient cells leads to mitochondrial localization, preceding apoptosis. Overexpression of anti-apoptotic Bcl-2 or Bcl-xL blocks mitochondrial p53 accumulation and apoptosis but not cell cycle arrest, suggesting a feedback loop between p53 and mitochondrial apoptotic regulators. Targeting p53 to mitochondria using import leader fusions induces apoptosis in p53-deficient cells, indicating a direct signaling role of p53 at mitochondria. This model proposes that p53 can contribute to apoptosis by direct signaling at mitochondria, amplifying the transcription-dependent apoptosis of p53. The study highlights the importance of mitochondria in p53-mediated apoptosis and suggests a novel role for p53 in direct apoptotic signaling at mitochondria, which are a strategic point in the cell death cascade. The findings support a novel role of p53 in direct apoptotic signaling at mitochondria, enhancing its apoptotic potency. The study also discusses the functional significance of mitochondrial p53 accumulation in p53-mediated apoptosis, showing that mitochondrial p53 localization is specific to p53-dependent apoptosis and not during p53-independent apoptosis or cell cycle arrest. The data suggest a regulatory feedback signaling loop between p53 and Bcl-2 family members at the mitochondrial level. Targeting p53 to mitochondria is sufficient to induce apoptosis in p53-deficient cells, indicating that p53 can launch an apoptotic pathway from the mitochondrial level. The study concludes that p53 has a direct role in a novel apoptosis signaling pathway at the mitochondrion, acting in synergy with the transcription-dependent mode of action of p53, thereby amplifying its apoptotic potency.