The mechanisms involved in the metabolic changes induced by cold stress in isolated rat liver mitochondria were studied. Respiration, ATP synthesis, and membrane potential as well as the contents of several metabolites were determined in liver mitochondria from cold-exposed rats. At different times of cold exposure, the force–flux relationships showed net variation in flux (enhanced respiration, diminished ATP synthesis) with no associated variation in force (H⁺ gradient); this suggested that decoupling rather than classical uncoupling was involved in the effects of cold stress. The flux control coefficient of the H⁺ leak on basal respiration was slightly increased by 380 h of cold exposure. Cold stress also induced a diminution in total membrane fatty acids, Zn²⁺, Fe³⁺, ATP, and ADP/O ratios; the content of cytochromes c + c ₁ and b oscillated. The contents of Ca²⁺, Na⁺, P_i, and cytochromes a + a ₃ were not affected, whereas matrix ADP, AMP, K⁺, and Mg²⁺ were markedly increased. Basal and oleic acid-stimulated respiration of mitochondria from cold-stressed rats was inhibited by GDP, carboxyatractyloside, or albumin. These agents did not affect basal respiration in control mitochondria. Western blot analysis showed enhanced expression of a protein of about 35 kDa, presumably the uncoupling protein 2, induced by long-term cold exposure. The overall data suggest that cold stress promoted decoupling of oxidative phosphorylation, and hence, changes in several matrix metabolites, by increasing free fatty acids and the UCP2 content.