Bcl-xS, a pro-apoptotic member of the Bcl-2 protein family, is localized in the mitochondrial outer membrane and induces caspase-dependent and nerve growth factor (NGF)-inhibitable apoptosis in PC12 cells. The mechanism of action of Bcl-xS and how NGF inhibits this death are not fully understood. It is still unknown whether Bcl-xS induces mitochondrial cytochrome c release, and which apoptotic step NGF inhibits. We show that Bcl-xS induces cytochrome c release and caspase-3 activation in several cell types, and that in PC12 cells, these events are inhibited by NGF treatment. The survival effect of NGF was inhibited by inhibitors of protein kinase C (PKC), phosphatidylinositol-3-kinase (PI 3-kinase), and the mitogen-activated protein kinase kinase (MEK) inhibitors GF109203X, LY294002, and U0126. These findings show that cytochrome c release and caspase-3 activation participate in Bcl-xS-induced apoptosis, and that NGF inhibits Bcl-xS-induced apoptosis at the mitochondrial level via the PKC, PI 3-kinase, and MEK signaling pathways.