Impairment of the normal spatiotemporal pattern of intracellular Ca²⁺ ([Ca²⁺]_i) signaling, and in particular, the transition to an irreversible “Ca²⁺ overload” response, has been implicated in various pathophysiological states. In some diseases, including pancreatitis, oxidative stress has been suggested to mediate this Ca²⁺ overload and the associated cell injury. We have previously demonstrated that oxidative stress with hydrogen peroxide (H₂O₂) evokes a Ca²⁺ overload response and inhibition of plasma membrane Ca²⁺-ATPase (PMCA) in rat pancreatic acinar cells (Bruce JI and Elliott AC. Am J Physiol Cell Physiol 293: C938–C950, 2007). The aim of the present study was to further examine this oxidant-impaired inhibition of the PMCA, focusing on the role of the mitochondria. Using a [Ca²⁺]_i clearance assay in which mitochondrial Ca²⁺ uptake was blocked with Ru-360, H₂O₂ (50 μM–1 mM) markedly inhibited the PMCA activity. This H₂O₂-induced inhibition of the PMCA correlated with mitochondrial depolarization (assessed using tetramethylrhodamine methylester fluorescence) but could occur without significant ATP depletion (assessed using Magnesium Green fluorescence). The H₂O₂-induced PMCA inhibition was sensitive to the mitochondrial permeability transition pore (mPTP) inhibitors, cyclosporin-A and bongkrekic acid. These data suggest that oxidant-induced opening of the mPTP and mitochondrial depolarization may lead to an inhibition of the PMCA that is independent of mitochondrial Ca²⁺ handling and ATP depletion, and we speculate that this may involve the release of a mitochondrial factor. Such a phenomenon may be responsible for the Ca²⁺ overload response, and for the transition between apoptotic and necrotic cell death thought to be important in many disease states.