During late gestation, amino acids and insulin promote skeletal muscle protein synthesis. However, the independent effects of amino acids and insulin on the regulation of mRNA translation initiation in the fetus are relatively unknown. The purpose of this study was to determine if an acute amino acid infusion in the late gestation ovine fetus, with and without a simultaneous increase in fetal insulin concentration, activates translation initiation pathway(s) in skeletal muscle. Fetuses received saline (C), mixed amino acid infusion plus somatostatin infusion to suppress amino acid-stimulated fetal insulin secretion (AA+S), mixed amino acid infusion with concomitant physiological increase in fetal insulin (AA), or high dose insulin infusion with euglycemia and euaminoacidemia (HI). After a 2-hour infusion period, fetal skeletal muscle was harvested under in vivo steady-state conditions and frozen for quantification of proteins both up-stream and down-stream of mTOR. In the AA group, we found a 3-fold increase in p70S6K and Erk 1/2 phosphorylation; however, blocking the physiological rise in insulin with somatostatin in the AA+S group prevented this increase. In the HI group, Akt, Erk 1/2, p70S6K, and ribosomal protein S6 were highly phosphorylated and 4E-BP1 associated with eIF4E decreased by 30%. These data show that insulin is a significant regulator of intermediates involved in translation initiation in ovine fetal skeletal muscle. Furthermore, the effect of amino acids is dependent on a concomitant increase in fetal insulin concentrations, as amino acid infusion up-regulates p70S6K and Erk only when amino acid-stimulated increase in insulin occurs.