Evidence suggests that p53 induces cell death by a dual mode of action involving activation of target genes and transcriptionally independent direct signaling. Mitochondria are major signal transducers in apoptosis. We recently discovered that a fraction of induced p53 protein rapidly translocates to mitochondria during p53-dependent apoptosis, but not during p53-independent apoptosis or p53-mediated cell cycle arrest. Importantly, specific targeting of p53 to mitochondria was sufficient to induce apoptosis in p53-deficient tumor cells. This led us to propose a model where p53 exerts a direct apoptogenic role at the mitochondria, thereby enhancing the transcription-dependent apoptosis of p53. Here we show for the first time that mitochondrial localization of endogenous p53 can be visualized by immunofluorescence of whole cells when stressed by hypoxic conditions. Suborganellar localization by limited trypsin digestion of isolated mitochondria from stressed cells suggests that a significant amount of mitochondrial p53 is located at the surface of the organelle. This mitochondrial association can be reproduced in vitro with purified p53. Together, our data provide further evidence for an apoptogenic signaling role of p53 protein in vivo at the level of the mitochondria.