In vitro experiments have demonstrated independent, additive casein synthesis responses to supplies of Ile, Leu, Met, and Thr. We hypothesized that lactating cattle would respond in a similar manner. Forty-eight Holstein cows were fed a diet containing 75% of NRC (2001) predicted, metabolizable protein (MP) requirements (LoMP, 13.5% CP) in a randomized block design with replicated 4 × 4 Latin squares within each block. Each of the 4 ruminally protected (RP) amino acids (AA) represented a block. Period length was 12 d. Treatments within each block were LoMP and LoMP plus RPIle, RPLeu, RPMet, or RPThr at doses of 0, 50, 100, and 150% of the difference between absorbed AA supplied by the LoMP and MP sufficient diets. Intestinal availability of each RPAA was assessed by abomasal dosing of the RPAA after 8 h of ruminal incubation. The RPAA doses were 0, 8.5, 17, and 25.5 g of absorbed Ile/cow/d; 0, 14, 28, and 42 g of absorbed Leu/cow/d; 0, 3, 6, and 9 g of absorbed Met/cow/d; and 0, 8, 16, and 24 g of absorbed Thr/cow/d. DMI increased linearly with increasing dose of Ile (P = 0.02), and tended to increase quadratically with respect to Met and Thr. Leu had no effect on DMI. Milk yield (kg/d) increased quadratically (P < 0.05) in response to Ile, Met, and Thr, and decreased quadratically in response to Leu. Milk protein yield (kg/d) tended to increase quadratically (P = 0.11) in response to Met and linearly (P = 0.12) in response to Thr, and decreased quadratically in response to Leu. Ile had no effect. Body weight (kg/d) decreased quadratically (P < 0.0001) with Met dose, and tended to increase linearly (P = 0.11) with Leu dose, suggesting that changes in milk protein yield for animals supplemented with Leu may be driven by non-mammary tissue use. Conversely, DMI and BW responses for animals supplemented with Met and Thr do not explain the trend for increased milk protein yield, suggesting that Met and Thr stimulated milk protein synthesis. Revising dairy requirement models to include animal responses to individual AA may improve milk production predictions leading to increased N efficiency, and reduced N excretion from lactating dairy animals.