Five Finnish ruminally cannulated Ayrshire cows were used in a 5 x 5 Latin square trial with 14-d periods to determine whether branched-chain amino acids (AA) are the second- or colimiting AA for milk protein synthesis on grass silage-cereal based diet. Mammary metabolism of AA as well as AA supply from the basal diet were also studied. Grass silage (17.5% crude protein) was given ad libitum with 9 kg/d as a cereal-based concentrate (13.8% crude protein). Treatments were basal diet without AA infusion (Control), abomasal infusion of AA mixture of His, Ile, Leu, and Val at 8.5, 14.9,27.9, and 18.3 g/d, respectively, AA mixture minus Ile, AA mixture minus Leu, and AA mixture minus Val. Glucose was infused on all treatments at 250 g/d. Amino acid infusions had no effect on dry matter intake (mean 19.2 kg/d), yields of milk (mean 25.3 kg/d), energy-corrected milk (mean 25.9 kg/d), milk protein (mean 807 g/d), lactose (mean 1261 g/d), or fat (mean 1056 g/d). Milk composition was not affected by the treatments. Plasma concentrations of His and Val responded to AA infusions but concentration of Ile increased only on treatment AA mixture minus Leu, and concentration of Leu only on treatment AA mixture minus Ile. Infusion of AA mixture of His, Ile, Leu, and Val decreased plasma concentrations of Arg, Lys, Met, Phe, and Tyr. Amino acid infusions did not affect concentrations of plasma urea and energy metabolites or AA utilization by the mammary gland. Based on unchanged production parameters, the supply of His or branched-chain AA seemed not to be limiting under the current dietary conditions. Changes in plasma AA concentrations suggest either antagonism between individual AA in absorption or increased partitioning of AA into the muscle tissues. About 75% of omasal canal nonammonia nitrogen flow (427 g/d) was of microbial origin, and AA profiles of microbial protein and omasal canal digesta were fairly similar. Postruminal AA supply seems to be dependent on the basal diet, but variation may exist even within the similar basal diets.