This study examined the actions of 17β-estradiol (E2) and progesterone on the regulation of the peroxisome proliferator-activated receptors (PPARα and PPARγ) family of nuclear transcription factors and the mRNA abundance of key enzymes involved in fat oxidation, in skeletal muscle. Specifically, carnitine palmitoyltransferase I (CPT I), β-3-hydroxyacyl CoA dehydrogenase (P-HAD), and pyruvate dehydrogenase kinase 4 (PDK4) were examined. Sprague-Dawley rats were ovariectomized and treated with placebo (Ovx), E2, progesterone, or both hormones in combination (E+P). Additionally, sham-operated rats were treated with placebo (Sham) to serve as controls. Hormone (or vehicle only) delivery was via time release pellets inserted at the time of surgery, 15 days prior to analysis. E2 treatment increased PPARα mRNA expression and protein content (P<0.05), compared with Ovx treatment. E2 also resulted in upregulated mRNA of CPT I and PDK4 (P<0.05), PPARγ mRNA expression was also increased (P<0.05) by E2 treatment, although protein content remained unaltered. These data demonstrate the novel regulation of E2 on PPARα and genes encoding key proteins that are pivotal in regulating skeletal muscle lipid oxidative flux.