Arsenate (AsV) is a structural analogue of phosphate (P_i), yet its toxic effect is likely due to its reduction to the more toxic arsenite (AsIII), the mechanism of which is still unclear. Since mitochondria take up AsV as they do P_i, they may reduce AsV to AsIII. To test this hypothesis isolated rat liver mitochondria were incubated with AsV, and the incubate was analyzed for AsV and AsIII by HPLC–HG–AFS. Mitochondria rapidly reduced AsV to AsIII. Of the substrates supporting the citric acid cycle, glutamate enhanced the reduction most effectively. ADP increased, whereas AMP and ATP decreased, AsIII formation. These effects could be prevented by atractyloside. Electron transport inhibitors and uncouplers abolished AsIII formation, whereas ATP-synthase inhibitors almost completely inhibited it. Phosphate decreased AsIII formation in a concentration-dependent manner. Inhibitors of mitochondrial P_i-moving transporters abolished AsIII formation. Sulfate, sulfite, or thiosulfate that are transported by the dicarboxylate carrier caused partial inhibition. AsIII was recovered completely from the supernatant of the mitochondrial incubate, suggesting that mitochondria exported the formed AsIII. Testing the effects on mitochondrial AsV reduction of chemicals that are inhibitors or substrates of thioredoxin reductase failed to support the role of this enzyme in reduction of AsV. Depletion of mitochondrial glutathione impaired mitochondrial AsV reducing activity but also diminished the respiratory control ratio. Upon solubilization of mitochondria, their AsV-reducing activity was lost and was not recovered by addition of GSH and NADH or NADPH. Summarizing, mitochondria work as reactors: they take up AsV, rapidly reduce it, and export the formed AsIII. Disruption of functional or structural integrity of mitochondria severely impairs biotransformation of AsV into AsIII in this organelle.