Effects of providing a limiting AA on milk secretion and mammary nutrient uptake were investigated using four lactating dairy cows given duodenal infusions of graded amounts of L-Lys.HCl (0, 9, 27, and 63 g/d). Infusions were administered over 4-d periods according to a Latin square design. Diets consisted of a 70:30 corn silage:concentrate ratio supplemented with 1.5 kg of DM/d as dehydrated alfalfa. The basal diet met requirements for energy and protein and was deficient in Lys. Cows were fitted with indwelling catheters inserted into the left carotid and left subcutaneous abdominal veins, and a transit-time flow probe was implanted around the left external pudic artery. Milk, fat, and protein yields were unaffected by infusions of Lys. Milk protein content increased to a maximum with the third treatment, and fat content showed opposite variation. Although arterial concentrations and arteriovenous differences of Lys increased to a plateau with the third treatment, the relationship between arteriovenous differences and arterial concentrations was curvilinear. Infusions of Lys tended to increase the efficiency of N utilization and the mammary extraction rates of AA to synthesize more milk proteins. Initially, Lys was extracted in a direct ratio to its milk output and then taken up in excess by the secretory cells, suggesting that Lys was no longer a limiting factor in milk protein synthesis.