Treatment of bovine pulmonary artery smooth muscle mitochondria with the calcium ionophore, A23187 (0.2 μM) stimulates μ-calpain activity and subsequently cleaves Na+/Ca2+ exchanger (NCX). Pretreatment of the A23187 treated mitochondria with the calpain inhibitors, calpeptin or MDL28170 or with Ca2+ chelator, EGTA does not cleave NCX. Treatment of the mitochondria with A23187 increases Ca2+ level in the mitochondria, which subsequently dissociates μ-calpain-calpastatin association leading to the activation of μ-calpain. Immunoblot study of the A23187 treated mitochondria with the NCX polyclonal antibody indicates the degradation of mitochondrial inner membrane NCX (110 kDa) resulting in the doublet of ∼54-56 kDa NCX fragments. Moreover, in vitro cleavage of mitochondrial purified NCX by mitochondrial purified μ-calpain supports our conclusion. This cleavage of NCX may be interpreted as the main cause of Ca2+ overload and could lay a key role in the activation of apoptotic process in pulmonary smooth muscle. © 2008 Elsevier Inc. All rights reserved.