Four high-yielding lactating Holstein cows fitted with duodenal cannulas were used in the experiment. Cows were divided into 2 groups -control (Control) with leucine deficiency and experimental (Leucine) with a leucine supplement. The experiment was divided into 4 periods of 7 days, each consisting of a 3-day preliminary period followed by a 4-day experimental period. In the first period, 2 cows were assigned to Control and the remaining 2 to Leucine. In the subsequent period the cows were switched to the other treatment. Cows were fed individually twice daily the basal diet based on maize silage, lucerne hay and supplemental mixture. Infusions of amino acids in Leucine consisted of methionine (12.6 g/day), lysine (20.7 g/day), histidine (10.7 g/day) and leucine (19.3 g/day). The composition of amino acid infusate in Control was the same except for leucine that was replaced with monosodium L-glutamate. The intake of dry matter was not affected by the treatment (P > 0.05). No effect of leucine infusion on milk yield and composition was observed (P > 0.05), nevertheless the concentration of protein and casein in milk tended to be higher in Leucine (38.3 and 31.3 g/kg) than in Control (37.4 and 30.4 g/kg, respectively, P < 0.1). The yield of milk components was not affected by the treatment (P > 0.05). Duodenal infusion of leucine resulted in a decreased plasma level of isoleucine in Leucine compared to Control (P < 0.01). Concentrations of leucine, cysteine and citrulline tended to be higher and the concentration of tyrosine tended to be lower in Leucine in comparison with Control (P < 0.10).