We identified recently the p53AIP1 gene, a novel p53 target that mediates p53-dependent apoptosis. In the experiments reported here, ectopic expression of p53AIP1 induced down-regulation of mitochondrial DeltaPsim and release of cytochrome c from mitochondria in human cells. Immunoprecipitation and immunostaining experiments indicated interaction between p53AIP1 and bcl-2 proteins at mitochondria. Overexpression of bcl-2 blocked the down-regulation of mitochondrial DeltaPsim and the proapoptotic activity of p53AIP1. Our results implicate p53AIP1 as a pivotal mediator of the p53-dependent mitochondrial apoptotic pathway.