Calcium-dependent cysteine proteases, calpains, have physiological roles in cell motility and differentiation but also play a pathological role following insult or disease. The ubiquitous calpains are widely considered to be cytosolic enzymes, although there has been speculation of a mitochondrial calpain. Within a highly enriched fraction of mitochondria obtained from rat cortex and SH-SY5Y human neuroblastoma cells, immunoblotting demonstrated enrichment of the 80 kDa mu-calpain large subunit and 28 kDa small subunit. In rat cortex, antibodies against domains II and III of the large mu-calpain subunit also detected a 40 kDa fragment, similar to the autolytic fragment generated following incubation of human erythrocyte mu-calpain with Ca2+. Mitochondrial proteins including apoptosis inducing factor and mitochondrial Bax are calpain substrates, but the mechanism by which calpains gain access to these proteins is uncertain. Mitochondrial localization of mu-calpain places the enzyme in proximity to its mitochondrial substrates and to Ca2+ released from mitochondrial stores. (c) 2005 Elsevier Inc. All rights reserved.