The mechanisms of Ca2+-induced release of Cytochrome c (Cyt c) from rat brain mitochondria were examined quantitatively using a capture ELISA. In 75 or 125 mM KCI-based media 1.4 μmol Ca2+/mg protein caused depolarization and mitochondrial swelling. However, this resulted in partial Cyt c release only in 75 mM KCI. The release was inhibited by Ru360, an inhibitor of the Ca2+ uniporter, and by cyclosporin A plus ADP, a combination of mitochondrial permeability transition inhibitors. Transmission electron microscopy (TEM) revealed that Ca2+-induced swelling caused rupture of the outer membrane only in 75 mM KCI. Koenig's polyanion, an inhibitor of mitochondrial porin (VDAC), enhanced swelling and amplified Cyt c release. Dextran T70 that is known to enhance mitochondrial contact site formation did not prevent Cyt c release. Exposure of cultured cortical neurons to 500 μM glutamate for 5 min caused Cyt c release into the cytosol 30 min after glutamate removal. MK-801 or CsA inhibited this release. Thus, the release of Cyt c from CNS mitochondria induced by Ca2+ in vitro as well as in situ involved the mPT and appeared to require the rupture of the outer membrane.