Infusing glucose into cows increases milk protein yield in some experiments but not others. Invariably, glucose infusion decreases plasma concentrations of the branched-chain amino acids (BCAA) Val, Ile and Leu. The objective of this study was to evaluate the response to replenishment of BCAA during postruminal glucose infusion. Twelve cows (80 ± 22 DIM) were assigned, in a replicated 4 × 4 Latin square design, to 96-h continuous jugular infusions of saline, 1 kg/d glucose, or 1 kg/d glucose + 75 g/d or 150 g/d BCAA. All cows were given ad libitum access to a TMR of 12.6% crude protein and 1.52 Mcal/kg NE_L on a dry basis. Infusion of glucose alone did not affect DMI, milk yield, or protein yield (P > 0.19), but increased lactose yield 98 g/d compared with saline (P = 0.05). Addition of BCAA to glucose infusions caused a linear decrease in milk protein yield (P < 0.01), and tended to decrease DMI, milk yield, and lactose yield (P < 0.09). Concentrations of non-branched-chain essential amino acids (non-BEAA) in plasma decreased 19% (P < 0.01) during glucose infusion and BCAA concentrations decreased 30% (P < 0.01). Mammary blood flow was 30% higher (P < 0.01) during glucose infusion and net mammary uptakes of essential AA remained unchanged compared with saline (P > 0.24). Concentrations of BCAA in plasma returned to 6% higher (P = 0.34) than levels on the saline control at the BCAA infusion rate of 75 g/d and were 49% higher than control levels (P < 0.01) at 150 g/d of infusion. Addition of BCAA to glucose infusions caused a linear decline in non-BEAA concentrations in plasma (P < 0.01), as well as their mammary uptakes (P = 0.04). Plasma urea concentration was unaffected by BCAA infusion (P = 0.88), indicating that catabolism of non-BEAA was not stimulated. Evidence that neither mammary utilization nor whole-body catabolism of non-BEAA accounted for their disappearance from plasma leads us to conclude that BCAA caused a partitioning of non-BEAA to extra-mammary tissues for protein deposition. It was estimated that 60 g/d BCAA was sufficient to counteract the decrease in plasma BCAA concentrations induced by 1 kg/d i.v. glucose.