Dairy cows require amino acids (AA) in the correct amount and profile to maximize milk protein secretion. This study was conducted to examine the effects of a deficiency of 3 essential AA - phenylalanine (Phe), threonine (Thr) and tryptophan (Trp) - on mammary metabolism and milk production. Five Holstein cows were abomasally infused with water or AA solutions that contained all AA, or were deficient in Phe, Thr, or Trp. Milk and milk protein yields decreased with a deficiency of Phe or Thr but not Trp. Arterial concentrations of Phe, Thr, and Trp decreased with their respective deletions, whereas concentrations of metabolites that serve as energy substrates were unaffected by treatment. Mammary plasma flow and mammary uptake of several AA changed in response to treatments, demonstrating that the mammary gland can respond to changes in nutrient supply in an attempt to maximize milk synthesis.