Exposures of biological systems to increased concentrations of reactive oxygen species frequently are characterized by measurements of glutathione disulfide (GSSG), and such increases are cited as criteria for oxidant stresses. Evidence suggests that some oxidant stresses are compartmentalized in the mitochondria, but efforts to test these hypotheses on experimental models of toxicant challenge in vivo are severely limited by the inability to prevent redox reactions and thiol-disulfide exchange during isolation of mitochondria. Coenzyme A (CoASH) is localized predominantly in mitochondria and exchanges with GSH/GSSG. CoASH and CoASSG can be measured by HPLC, which suggests that assessment of CoASH/CoASSG contents in tissue samples freeze clamped immediately upon removal of the tissue and processed in acidic buffers to minimize chemical alterations during sample processing might provide critical information regarding intramitochondrial thiol redox status in relevant experimental models. Male mice were treated with 400 mg/kg of furosemide in saline or equal volumes of saline and killed 2 or 6 h later, their livers freeze-clamped, and levels of GSH, GSSG, CoASH and CoASSG were measured in acid supernatants. Increases in plasma ALT activities documented hepatic injury. Hepatic GSH and GSSG levels were not different in the furosemide-treated mice, indicating no oxidant stress in the larger cytosolic compartment. CoASH contents in the furosemide-treated animals were not different from the controls (112±14 nmol/g tissue) at either 2 or 6 h. This observation indicates that decreases in tissue CoASH contents are not obligatory consequences of cell death in vivo. Hepatic CoASSG levels were significantly higher at 2, 4 and 6 h (two-way ANOVA, P<0.009) in the furosemide-treated animals (7.3±1.5, 5.1±0.8, 8.6±1.4; vs. 3.5±0.5, 4.4±0.6, 4.5±0.6 nmol/g of tissue in controls). CoASH and CoASSG levels measured in acid supernatants of mitochondria isolated by differential centrifugation of homogenates of unfrozen tissues were not different. Pellets from acid-precipitated mitochondrial proteins were treated with dithioerythritol (DTE) and CoASH released from CoASSP measured. No differences in CoASSP were observed in furosemide-treated animals. These results indicate an unexpected oxidant stress response to furosemide that appears to be expressed preferentially in the mitochondrial compartment.