Poly(ADP-ribose) polymerase-1 (PARP-1) mediates neuronal cell death in a variety of pathological conditions involving severe DNA damage. Poly(ADP-ribose) (PAR) polymer is a product synthesized by PARP-1. Previous studies suggest that PAR polymer heralds mitochondrial apoptosis-inducing factor (AIF) release and thereby, signals neuronal cell death. However, the details of the effects of PAR polymer on mitochondria remain to be elucidated. Here we report the effects of PAR polymer on mitochondria in cells in situ and isolated brain mitochondria in vitro. We found that PAR polymer causes depolarization of mitochondrial membrane potential and opening of the mitochondrial permeability transition pore early after injury. Furthermore, PAR polymer specifically induces AIF release, but not cytochrome c from isolated brain mitochondria. These data suggest PAR polymer as an endogenous mitochondrial toxin and will further our understanding of the PARP-1-dependent neuronal cell death paradigm.