In the present study, we have investigated gender differences in rat liver mitochondrial oxidative metabolism. Total mitochondrial population (M) as well as the heavy (M1), medium (M3), and light (M8) mitochondrial fractions obtained by means of differential centrifugation steps at 1,000, 3,000, and 8,000 g, respectively, were isolated. Electron microscopic analysis was performed and mitochondrial protein content and cardiolipin levels, mitochondrial O(2) flux, ATP synthase activity, mitochondrial membrane potential, and mitochondrial transcription factor A (TFAM) protein levels were measured in each sample. Our results indicate that mitochondria from females have higher protein content and higher cardiolipin levels, greater respiratory and phosphorylative capacities, and more-energized mitochondria in respiratory state 3. Moreover, protein levels of TFAM were four times greater in females than in males. Gender differences in the aforementioned parameters were more patent in the isolated heavy M1 and M3 mitochondrial fractions. The present study demonstrates that gender-related differences in liver mitochondrial function are due mainly to a higher capacity and efficiency of substrate oxidation, likely related to greater mitochondrial machinery in females than in males, which is in accord with greater mitochondrial differentiation in females.