The study investigates the role of p53 protein in apoptosis, focusing on its localization to mitochondria. It is found that a fraction of p53 protein localizes to mitochondria during p53-dependent apoptosis but not during p53-independent apoptosis or cell cycle arrest. This localization is rapid and precedes changes in mitochondrial membrane potential, cytochrome c release, and procaspase-3 activation. The majority of mitochondrial p53 localizes to the membranous compartment, while a fraction is found in a complex with the mitochondrial import motor mt hsp70. Overexpression of anti-apoptotic Bcl-2 or Bcl-xL abrogates stress signal-mediated mitochondrial p53 accumulation and apoptosis but not cell cycle arrest, suggesting a feedback signaling loop between p53 and mitochondrial apoptotic regulators. Importantly, targeting p53 to mitochondria using import leader fusions is sufficient to induce apoptosis in p53-deficient cells. The findings support a novel role for p53 in direct apoptotic signaling at mitochondria, which amplifies the transcription-dependent apoptosis of p53.The study investigates the role of p53 protein in apoptosis, focusing on its localization to mitochondria. It is found that a fraction of p53 protein localizes to mitochondria during p53-dependent apoptosis but not during p53-independent apoptosis or cell cycle arrest. This localization is rapid and precedes changes in mitochondrial membrane potential, cytochrome c release, and procaspase-3 activation. The majority of mitochondrial p53 localizes to the membranous compartment, while a fraction is found in a complex with the mitochondrial import motor mt hsp70. Overexpression of anti-apoptotic Bcl-2 or Bcl-xL abrogates stress signal-mediated mitochondrial p53 accumulation and apoptosis but not cell cycle arrest, suggesting a feedback signaling loop between p53 and mitochondrial apoptotic regulators. Importantly, targeting p53 to mitochondria using import leader fusions is sufficient to induce apoptosis in p53-deficient cells. The findings support a novel role for p53 in direct apoptotic signaling at mitochondria, which amplifies the transcription-dependent apoptosis of p53.