Higher fat and lower carbohydrate and amino acid oxidation are observed in women compared to men during endurance exercise. We hypothesized that the observed sex difference is due to estrogen, and menstrual cycle phase or supplementation of men with 17-estradiol (E2) would coordinately influence the mRNA content of genes involved in lipid and/or carbohydrate metabolism in skeletal muscle. Twelve men and women had muscle biopsies taken before and immediately after 90 min of cycling at 65% VO_2peak. Women were studied in the mid-follicular (FOL) and mid-luteal (LUT) phases and men were studied after 8 d of E2 or placebo supplementation. Targeted RT-PCR was used to compare mRNA content for genes involved in transcriptional regulation, lipid, carbohydrate, and amino acid metabolism. Sex was the greatest predictor of substrate metabolism gene content. Sex affected the mRNA content of FATm, FABPc, SREBP-1c, mtGPAT, PPAR, PPAR, CPTI, TFP, GLUT4, HKII, PFK and BCOADK (P < 0.05). E2 administration significantly (P < 0.05) affected the mRNA content of PGC-1, PPAR, TFP, CPT1, SREBP-1c, mtGPAT, GLUT4, GS1 and AST. Acute exercise increased the mRNA abundance for PGC-1, HSL, FABP_c, CPTI, GLUT4, HK II and AST (P < 0.05). Menstrual cycle had a small effect on PPAR, GP and glycogenin mRNA content. Overall, women have greater mRNA content for several genes involved in lipid metabolism that is partially due to an effect of 17-estradiol.