Leucine and to a lesser extent, isoleucine, possess signaling properties promoting cellular anabolic metabolism via mammalian target of rapamycin, but in vivo evidence is limited. In contrast, methionine, lysine, and histidine are well documented to limit milk protein synthesis for some diets. The objective of this study was to evaluate production responses from supplementing 2 groups of amino acids in a 2 × 2 factorial design. Eight cows (102 ± 19 DIM) were assigned to 4 jugular infusion treatments consisting of saline (SAL), 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 amino acid 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. The SAL diet had observed daily deficits of 539 g of metabolizable protein, 20 g of methionine, 48 g of lysine, 7 g of histidine, 89 g of leucine, and 25 g of isoleucine according to CNCPS v6.55 and 498 g of metabolizable protein by NRC (2001). Cows were ad libitum fed a common diet consisting of 15.2% crude protein, and 1.61 Mcal/kg NE_L on a dry basis. Dry matter intake increased for MKH by 0.5 kg/d (P = 0.05). Milk yield increased 2.3 kg/d (P = 0.02) by infused IL and no change was observed for MKH (P = 0.39). Milk protein concentration increased by 0.13 percentage units for MKH (P < 0.001) whereas yield increased for both MKH and IL by 88 g/d (P < 0.01) and 57 g/d (P = 0.02) respectively. The milk protein yield increase for MKH⁺IL was 145 g/d (P < 0.01) versus SAL. Energy corrected milk tended to increase for IL by 1.6 kg/d (P = 0.13). No differences were observed for fat yield or concentrations, milk urea nitrogen, feed efficiency, body weight change, or milk N efficiency, that latter being 38.6%. 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 nutrient will limit milk protein production.