Essential AA (EAA), particularly leucine, isoleucine, methionine, and histidine, possess signaling properties for promoting cellular anabolic metabolism, whereas methionine, lysine, and histidine are considered also to be substrate limiting AA. The objective of this study was to evaluate production responses to supplementation of 2 AA groups in a 2 × 2 factorial design. Eight cows (99 ± 18 days in milk) were assigned to 4 jugular infusion treatments consisting of saline (CON), methionine plus lysine plus histidine (MKH), isoleucine plus leucine (IL), or MKH plus IL, in a replicated 4 × 4 Latin square design. Periods were 18 d in length, comprising 8 d of rest followed by 10 d of jugular infusion. Daily infusion amounts were 21 g of methionine, 38 g of lysine, 20 g of histidine, 50 g of leucine, and 22 g of isoleucine. Cows were ad libitum fed a common diet consisting of 15.2% crude protein and 1.61 Mcal/kg NE L on a dry matter basis that was predicted to meet rumen degradable protein requirements but was 15% deficient in metabolizable protein. Milk and energy-corrected milk yields increased by 2.3 kg/d and 1.9 kg/d, respectively, with infused IL, and no change was observed for MKH. Milk protein concentration increased by 0.13 percentage units for MKH, whereas milk protein yield increased for both MKH and IL by 84 g/d and 64 g/d, respectively. The milk protein yield increase for MKH + IL was 145 g/d versus CON. Gross feed efficiency tended to increase with IL infusion, and N efficiency tended to increase with MKH infusion. Aggregate arterial EAA concentrations less Met, Lys, and His declined by 7.2% in response to MKH infusion. Arterial EAA less Ile and Leu also declined by 6.2% in response to IL infusion. Net total AA (TAA) and EAA uptake by the udder tended to increase in response to MKH infusion, whereas mammary blood flow increased in response to IL infusion, but TAA and EAA net uptakes were unaffected. Apparent udder affinity increased for TAA and EAA less Met, Lys, and His in response to MKH infusion, whereas affinity for EAA less Ile and Leu increased for IL infusion. Venous Met and Leu concentrations increased by 192% and 35% from the MKH and IL infusions, respectively, compared with CON, which indicates that intracellular concentration of these EAA changed substantially. Increases in milk protein yield were observed from 2 groups of amino acids independently and additively, which contradicts the single limiting amino acid theory that a single EAA will limit milk protein yield.