Canola meal (CM) or by-products of ethanol production (dried distillers grain, DDG) may offer an economical alternative to soybean meal (SBM) in North American dairy rations. These protein supplements can effectively replace SBM and, in 2 recent meta-analyses, CM had a positive effect on milk and milk protein yields compared with SBM. The objective of this study was to determine if the positive responses observed with inclusion of CM in dairy rations could be explained by an increased availability of His, Lys, Met, or glucose. Eight Holstein dairy cows were used in a replicated 4×4 Latin square with 14-d periods. Cows were fed isonitrogenous (17.2% crude protein) and isoenergetic (1.56Mcal/kg of net energy of lactation) diets formulated to slightly exceed nutrient requirements. Diets contained 38% grass hay and 62% corn-based concentrate including SBM, CM, corn high-protein DDG (HPDDG), or wheat DDG plus solubles (WDDGS) as the single protein supplement. The effect of protein supplements on availability of His, Lys, Met, and glucose was estimated using variations in the whole-body (WB) flux of these nutrients, determined by isotopic dilution. As planned, dry matter intake and milk and milk protein yields were not affected by treatments and averaged 23.7, 31.4, and 1.14kg/d, respectively. Lactose yield did not differ among diets although milk lactose content tended to be lower with CM and WDDGS diets than with SBM and HPDDG diets. Lysine availability was affected by treatments: the highest WB irreversible loss rate (ILR) was observed for the CM diet (371g/d) and the lowest for HPDDG diet (290g/d); values for SBM and WDDGS were intermediate (330 and 316g/d, respectively). Availability of His and Met did not vary among diets and WB ILR averaged, respectively, 129 and 124g/d; the CM diet, however, had numerically the highest His and Met ILR. Plasma concentrations of most of the essential AA were higher with the CM diet and lower with the HPDDG diet, the exception being Leu for which the concentration was highest for the HPDDG diet. Glucose WB rate of appearance was altered by diet, with the highest mean observed for SBM (3,036g/d) and the lowest for CM (2,795g/d); the 2 diets with the lowest WB glucose rate of appearance (CM and WDDGS) also had the lowest dietary starch concentration. Overall, this study suggested that positive responses in milk and milk protein yields observed with inclusion of CM in dairy rations could be linked to a greater supply of metabolizable protein, including some essential AA, especially His, Lys, and Met, as glucose availability was certainly not increased in cows fed the CM diet.