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This trial aimed to evaluate mammary gland amino acid (AA) flux in response to hyperinsulinemia with two levels of dietary metabolizable protein (MP). Six Holstein cows were randomly assigned in a 2×2 factorial arrangement crossover design (two MP intake levels and two blood insulin concentrations). Insulin infusions were performed to achieve hyperinsulinemia while maintaining euglycemia. Arterial and venous blood was sampled every h over 8 h and the flux of each AA was calculated and expressed as mmol/l of milk. The MP intake increased the flux of Ile (P<0.01) and tended to increase the fluxes of Lys, Phe and Val (P≤0.10) from blood to mammary gland. Hyperinsulinemia increased the flux of Ala (P=0.01) and tended to increase the flux of Met (P=0.14). Lys tended to show an interaction among treatments.