Higher fat and lower carbohydrate and amino acid oxidation are observed in women compared with men during endurance exercise. We hypothesized that the observed sex difference is due to estrogen and that menstrual cycle phase or supplementation of men with 17β-estradiol (E2) would coordinately influence the mRNA content of genes involved in lipid and/or carbohydrate metabolism in skeletal muscle. Twelve men and twelve women had muscle biopsies taken before and immediately after 90 min of cycling at 65% peak oxygen consumption (V̇o2peak). Women were studied in the midfollicular (Fol) and midluteal (Lut) phases, and men were studied after 8 days of E2 or placebo supplementation. Targeted RT-PCR was used to compare mRNA content for genes involved in transcriptional regulation and 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, PPARδ, PPARα, CPTI, TFP-α, GLUT4, HKII, PFK, and BCOADK ( P < 0.05). E2 administration significantly ( P < 0.05) affected the mRNA content of PGC-1α, PPARα, PPARδ, TFP-α, CPTI, SREBP-1c, mtGPAT, GLUT4, GS-1, and AST. Acute exercise increased the mRNA abundance for PGC-1α, HSL, FABPc, CPTI, GLUT4, HKII, and AST ( P < 0.05). Menstrual cycle had a small effect on PPARδ, GP, and glycogenin mRNA content. Overall, women have greater mRNA content for several genes involved in lipid metabolism, which is partially due to an effect of E2.