This study was conducted to establish how AA supplied in a free form or as protein (casein, CN) affect the whole-body rate of appearance (WB Ra) of glucose, splanchnic and mammary glucose kinetics, and milk lactose secretion in lactating dairy cows. Five Holstein cows fitted with a rumen cannula and permanent indwelling catheters in the abomasum, portal, hepatic, and mesenteric veins, and one mesenteric artery, were used in a Youden square with 4 periods of 14 d each. Cows were fed a hay-based diet providing 100 and 70% of their net energy and metabolizable protein (MP) requirements, respectively. Treatments consisted of abomasal infusions of water (70% of MP requirements: control, Con), free AA (95% of MP requirements: AA1; and 120% of MP requirements: AA2), or CN (95% of MP requirements: CN1). The free AA mixture had the same profile as CN. On d 14 of each period, [6,6-2H2]glucose (25.8 mmol/h) was infused into a jugular vein, and blood samples (n=8) were taken over 4h from arterial, portal, hepatic, and mammary sources to measure glucose enrichment and concentration. Splanchnic and mammary plasma flows were determined by downstream dilution of para-aminohippurate and with the Fick principle, respectively. The last 6 milkings of each period were weighed and sampled to measure the yields of milk and components. The AA1 and CN1 treatments were not different for any of the measured parameters. Supplying AA linearly increased glucose WB Ra (AA2 vs. Con: +151 mmol/h) and liver net flux (+149 mmol/h). Utilization of glucose from the plasma compartment by the portal-drained viscera and liver and true portal absorption were not affected by AA supply. From these observations, we suggest that the increased WB Ra was due to increased net hepatic production. The AA from the infusion, in excess of that used to cover the increase in milk protein, were converted to glucose with an apparent efficiency close to 100% of maximum theoretical efficiency. Milk and lactose yields increased linearly with infusions of AA, by 14 and 16% with AA2 treatment, respectively. However, mammary glucose uptake was not significantly altered by AA infusions; this suggests that the mammary gland exerts active control on the uptake and utilization of glucose. For all treatments, the sum of true portal glucose absorption and true hepatic glucose production contributed more than 99% of WB Ra in the lactating cow; this would suggest that renal glucose synthesis makes only a small contribution to WB Ra under these conditions.