Mitochondrial membrane potential (⌬⌿ m ) depolarization contributes to cell death and electrical and contractile dysfunction in the post-ischemic heart. An imbalance between mitochondrial reactive oxygen species production and scavenging was previously implicated in the activation of an inner membrane anion channel (IMAC), distinct from the permeability transition pore (PTP), as the first response to metabolic stress in cardiomyocytes. The glutathione redox couple, GSH/GSSG, oscillated in parallel with ⌬⌿ m and the NADH/NAD ؉ redox state. Here we show that depletion of reduced glutathione is an alternative trigger of synchronized mitochondrial oscillation in cardiomyocytes and that intermediate GSH/GSSG ratios cause reversible ⌬⌿ m depolarization, although irreversible PTP activation is induced by extensive thiol oxidation. Mitochondrial dysfunction in response to diamide occurred in stages, progressing from oscillations in ⌬⌿ m to sustained depolarization, in association with depletion of GSH. Mitochondrial oscillations were abrogated by 4-chlorodiazepam, an IMAC inhibitor, whereas cyclosporin A was ineffective. In saponin-permeabilized cardiomyocytes, the thiol redox status was systematically clamped at GSH/GSSG ratios ranging from 300:1 to 20:1. At ratios of 150:1-100:1, ⌬⌿ m depolarized reversibly, and a matrix-localized fluorescent marker was retained; however, decreasing the GSH/GSSG to 50:1 irreversibly depolarized ⌬⌿ m and induced maximal rates of reactive oxygen species production, NAD(P)H oxidation, and loss of matrix constituents. Mitochondrial GSH sensitivity was altered by inhibiting either GSH uptake, the NADPH-dependent glutathione reductase, or the NADH/NADPH transhydrogenase, indicating that matrix GSH regeneration or replenishment was crucial. The results indicate that GSH/GSSG redox status governs the sequential opening of mitochondrial ion channels (IMAC before PTP) triggered by thiol oxidation in cardiomyocytes.The dual nature of oxygen as a vital electron acceptor in oxidative phosphorylation and as a dangerously reactive molecule has created pressure for the cell to evolve powerful antioxidant defenses that convert reactive oxygen species (ROS) 3 into harmless products to maintain a predominantly reduced redox environment. This depends on the following two factors: the reduction potential of the electron carriers, and the reducing capacity (i.e. the total concentration of the reduced species) of linked redox couples present in the cytoplasm or in the intraorganellar compartments (e.g. the mitochondrial matrix) of the cell. In addition to the redox couples involved in mitochondrial electron transport (the nicotinamide adenine dinucleotides NADH/NAD ϩ , and the flavins FADH 2 / FAD), the three main cellular redox pairs participating in intracellular reactions include reduced/oxidized glutathione (GSH/GSSG), thioredoxin (Trx(SH) 2 /TrxSS), and NADPH/ NADP ϩ , with the latter providing the thermodynamic driving force behind the glutathione and thioredoxin systems (1). The GSH/GSSG pool is the ...