Whole-body glucose rate of appearance (Ra) responses and milk lactose secretion were compared in dairy cows receiving duodenal infusions of glucose (Glc), a mixture of 5 nonessential amino acids (NEAAm), or ruminal infusions of propionic acid (C3). Four mid-lactation Holstein cows, fitted with both duodenum and rumen cannulas, were used in a 4 x 4 Latin square design with 14-d periods. Cows were fed a grass silage-based diet (Ctrl) that provided 88% of net energy of lactation and 122% of protein requirements. Concentrate was formulated with wheat (21.5%) and barley (20%) containing some starch. Isoenergetic infusions (5.15 Mcal/d of digestible energy) of Glc into the duodenum (7.7 mol/d), C3 into the rumen (14.1 mol/d), or NEAAm into the duodenum (in mol/d; Ala: 1.60; Asp: 0.60; Glu: 5.94; Gly: 1.22; Ser: 2.45) were given as a supplement to the Ctrl diet. During each period on d 13, [6,6-(2)H(2)] glucose was infused into one jugular vein and blood samples were taken from the other jugular vein to measure glucose enrichment and determine Ra. Dry matter intake decreased slightly with the infusions (6%), but did not differ among them. Whole body glucose Ra averaged 502, 745, 600, and 576 mmol/h for Ctrl, Glc, C3, and NEAAm, respectively. It increased with the increase in energy supply (Ctrl vs. infusions) and differed according to the nutrients infused. The Ra response was higher with Glc and C3 than with NEAAm and higher with Glc than with C3. Plasma concentrations of insulin were not affected, but insulin-like growth factor 1 increased with infusions. Plasma glucagon increased with NEAAm, which could favor the increased Ra. Overall, milk lactose yield (137, 141, 142, and 130 mmol/h for Ctrl, Glc, C3, and NEAAm, respectively) was not modified by the infusions, but was lower with NEAAm compared with Glc and C3. Changes in lactose yield did not parallel the increase in Ra, and therefore the ratio of lactose yield to Ra decreased with the infusions and was lower in Glc compared with C3, suggesting a shift of glucose utilization away from lactose synthesis toward other pathways, including mammary metabolism. Intestinal Glc was the most efficient nutrient in terms of increasing glucose Ra; however, there was no direct link between the increases in whole body glucose Ra observed with the 3 types of nutrients and milk lactose yield.