Sex differences in the cardiovascular system have been attributed to the effects of sex steroid hormones such as estrogen (E2), which are mainly mediated by their nuclear receptors ERα and ERβ. The goal of this study was to investigate the role of estrogen and their receptors on cardiac energy metabolism, in specific the influence on the oxidative phosphorylation.? We could show previously sex differences (SD) in the reponse of chronic pressure overload (PO) in cardiac enrergy metabolism. Gene expression profiling revealed that WT male hearts had a stronger repression of mitochondrial genes than WT female hearts after PO. Therefore we investigated the effects of sex and sexual hormones on the cardiac mitochondrial function in physiological conditions in skinned heart and skeletal muscles fibers. First, we could show in immunohistological analyses that estrogen receptors are present in cardiac mitochondria. Secondly, we performed treatment studies with E2 in an in vivo and ex vivo approach. WT mice from both sexes were treated once with an intraperitoneal injection of E2 (c=2.6µg/injection) for 5h and 24h. The mitochondrial activity was assessed in heart and muscles fibers using the Clark electrode. Maximal ADP stimulation and depletion (State III and IV) was recorded and the respiration control index (RCI) was calculated. State III respiration and RCI were enhanced in both sexes after E2 treatment at both time points. Notably, State III respiration and RCI was higher in males than in females after 24h. E2 treatment of skinned heart fibers ex vivo showed also an enhancement in the mitochondrial activity in both sexes after 24h and 48h, but not at earlier time points. Skeletal muscle fibers exhibited a higher state III respiration in both sexes, with a higher activity in females than in males.? This study shows that E2 enhances the activity of mitochondrial respiration accompanied with an increased oxidative phosphorylation in male and female mice. This observation might offer an explanation for SD observed in cardiac metabolism under PO conditions.