The effect of intestinal glucose supply on whole body rate of glucose appearance (WBGRa) and mammary utilization of glucose was studied in four lactating dairy cows. Glucose (0, 443, 963 and 2398 g/d) was continuously infused in the duodenum over 14-d periods using a Latin square design. A grass silage-based diet was formulated so that treatments were isoenergetic and isonitrogenous and contained 100 and 110% of energy and protein requirements according to INRA (1989). The WBGRa was measured by the [6,6-(2)H2]glucose dilution technique, and mammary glucose balance by arteriovenous differences and blood flow measurements. Duodenal glucose infusion increased arterial glucose concentrations linearly, whereas arterial concentrations of insulin, growth hormone, and glucagon were not changed. The WBGRa increased linearly with increasing glucose loads. The increase represented 42% of the intestinal glucose supplement. Mammary blood flow dramatically increased (up to 45%) and was associated with a significant increase of arterial insulin-like growth factor-1 concentrations. Mammary gland rate of glucose disappearance ([6,6-(2)H2]glucose measurement) increased linearly, whereas net mammary balance of glucose, lactose, and milk yields increased quadratically. Net mammary balance of glucose accounted for 60% of WBGRa, except for the greatest dose (47.6%). The decrease in milk yield with 2398 g/d of glucose may be explained by an imbalance in intracellular intermediate concentrations. The milk ratio of glucose-1-phosphate to glucose-6-phosphate decreased significantly at the greatest infusion of glucose. In conclusion, exogenous glucose supply to a grass silage-based diet increased WBGRa, mammary utilization of glucose and milk synthesis.