We previously reported that supplementing men with 17‐β‐estradiol (ES) reduces whole body leucine oxidation, improves estimated protein balance, decreases carbohydrate oxidation and increases lipid oxidation at rest and during exercise (Hamadeh et al, J Clin Endocrinol Metab 2005;90:3592–9.). We investigated the influence of menstrual phase, sex, estrogen supplementation and moderate intensity endurance exercise on skeletal muscle mRNA content of three genes involved in amino acid metabolism: aspartate aminotransferase (AST), branched‐chain α‐keto acid dehydrogenase (BCKD) and branched‐chain α‐keto acid dehydrogenase kinase (BCKDK), a deactivator of BCKD. Muscle biopsies from the vastus lateralis were obtained before and after 90 min of cycling at 65% VO2max in 12 women and 12 men. The women performed the exercise bout during the follicular (FOL, day 7–9) and luteal (LUT, day 19–21) phases. The men performed the exercise bout after eight days of ES (2 mg/d) and placebo (PL) supplementation, following a randomized, double‐blind, cross‐over design. Skeletal muscle mRNA content was measured using TaqMan® real time RT‐PCR. Menstrual phase and ES had no influence on the mRNA content of AST, BCKD and BCKDK. Sex had no influence on the mRNA content of AST and BCKD, however women had higher BCKDK mRNA content (FOL, 1.4 fold, P = 0.026; LUT, 1.3 fold, P = 0.05) as compared with PL men. Exercise had no influence on the mRNA content of BCKD and BCKDK, but increased that of AST by ~25% in PL men and women. We conclude that women have a lower capacity for branched‐chain amino acid breakdown (higher amounts of the inhibitory BCKDK), confirming our previous finding of a lower % activity of BCKD in women at rest. (Funded by the Hamilton Health Sciences Foundation and NSERC Canada).