The effects of temperature on the relationships among the rates of pyruvate carboxylation, O₂ uptake (J_o), oxidative phosphorylation (J_p), and the free energy of ATP hydrolysis (G_p) were studied in liver mitochondria isolated from 250-g female rats. Pyruvate carboxylation was evaluated at 37, 40, and 43°C. In disrupted mitochondria, pyruvate carboxylase maximal reaction velocity increased from 37 to 43°C with an apparent Q₁₀ of 2.25. A reduction in ATP/ADP ratio decreased enzyme activity at all three temperatures. In contrast, in intact mitochondria, increasing temperature failed to increase pyruvate carboxylation (malate + citrate accumulation) but did result in increased J_o and decreased extramitochondrial G_p. J_p was studied in respiring mitochondria at 37 and 43°C at various fractions of state 3 respiration, elicited with a glucose + hexokinase ADP-regenerating system. The relationship between J_o and G_p was similar at both temperatures. However, hyperthermia (43°C) reduced the J_p/J_o ratio, resulting in lower G_p for a given J_p. Fluorescent measurements of membrane phospholipid polarization revealed a transition in membrane order between 40 and 43°C, a finding consistent with increased membrane proton conductance. It is concluded that hyperthermia augments nonspecific proton leaking across the inner mitochondrial membrane, and the resultant degraded energy state offsets temperature stimulation of pyruvate carboxylase. As a consequence, at high temperatures approaching 43°C, the pyruvate carboxylation rate of intact liver mitochondria may fail to exhibit a Q₁₀ effect.