We studied effects of methylpyridinium ion (MPP+) on apoptosis, cell death and regulation of Bcl-2-family proteins in SH-SY5Y neuroblastoma cells. MPP+ increased intracellular accumulation of DNA-histone complexes as a measure of apoptosis and decreased intracellular calcein fluorescence as a measure of cell death. If ATP synthesis was supported, MPP+ caused apoptosis in ρ0 cells devoid of electron transport function. Caspase inhibition blocked apoptosis but not cell death caused by MPP+. MPP+ increased levels of Bax, Bcl-2 and Bcl-XL proteins ∼2-fold over 24 hr, with Bax increases occurring first; Bax did not increase in ρ0 cells. The Bax increase, but not that of Bcl-2 or Bcl-XL, was dependent on nitric oxide (NO) and seemed post-transcriptional. DAF-FM imaging revealed increased mitochondrial NO within hours of exposure to MPP+. Western blots showed a constitutive ∼130 kD protein that stained for NOS-2, consistent with reports of mitochondrial nitric oxide synthase (mtNOS). MPP+ caused a NO-dependent release of cytochrome C into cytoplasm. MPP+ increases mitochondrial NO levels and causes a NO-dependent increase in Bax protein, providing a mechanism for NOS-and Bax-dependency of MPTP neurotoxicity in vivo and implicating locally produced NO as a signaling molecule used by mitochondria to manipulate cell death cascades. © 2003 Wiley-Liss, Inc.