Four multiparous Holstein cows, each equipped with a duodenal cannula and an ultrasonic mammary blood flow probe, were assigned to a 4×4 Latin square to measure the effects of duodenal infusions of Leu (0, 40, 80 and 120 g/d) on lactational responses and mammary metabolism of nutrients. Cows were fed a diet of 67·0% corn silage, 5·3% grass hay, 14·2% peas, 7·8% maize starch, 1·0% each of molasses, urea, and sodium bicarbonate and 2·4% minerals and vitamins. Requirements of the remaining 9 essential amino acids were met by infusing into the duodenum 323 g/d of a mixture of amino acids including, Ile, Val, Met, Lys, Trp, Phe, His, Thr, Arg, Tyr and Glu. Milk protein content and yield were highest with 40 g/d of Leu and then progressively declined. Milk fat content and yield were significantly decreased over the entire range of Leu infusions. Arterial concentrations of Leu increased linearly and corresponding mammary arterio-venous differences increased to a plateau with 80 g/d of Leu. As Leu infusions increased, extraction rates of Leu decreased linearly, whereas those of Ile, Val, Lys, Arg, Thr and Tyr were significantly increased. Leu was taken up by the mammary gland according to milk output with 0 and 40 g/d of Leu infused and then largely in excess. Based on responses of protein yield and mammary uptake to milk output ratio, Leu concentration in total amino acids absorbed in the small intestine needs to be close to 8·9% for optimal milk protein synthesis.