The objective of this study was to determine whether dietary fat supplementation and level of undegradable intake protein (UIP) could affect daily milk output and composition by influencing nutrient supply to the mammary gland. Three lactating Holstein cows (60, 68, and 74 d postpartum) were used in an incomplete 4 x 4 Latin square design (2 x 2 factorial) and fed diets (15.9% CP and 19.5% ADF) with 0% or 2.5% partially hydrogenated tallow and 33% or 41% UIP. A 5:2.5:1 mixture of dried brewer's grains, corn gluten meal, and blood meal was substituted for soybean meal to raise dietary UIP from 33% to 41% UIP. Despite similar DM intake across treatments, cows produced 9% more milk per day when fed 2.5% supplemental fat, 41% UIP, or the combination of 2.5% fat and 41% UIP when compared to the control diet. Fat supplementation depressed milk protein percentage but not daily milk protein output. Mammary blood flow was estimated using the Fick principle at 6-hr intervals for 24 h. Concentration of individual nutrients in arterial (carotid) and venous (abdominal vein) blood and corresponding blood flows were used to calculate nutrient uptakes by the mammary gland. Calculated carbon uptake was 95 to 101% of output when using estimated carbon content of nutrients, and 100 to 106% when using an elemental analyzer to determine actual carbon output in milk. Uptake of glucose, β-hydroxybutyrate, lactate, pyruvate, acetate, and O₂ were not affected by dietary treatment. Triacylglycerol concentration in arterial blood and uptake of long-chain fatty acids were elevated by fat supplementation, resulting in milk fat with a higher percentage of 18-carbon fatty acids and a lower ratio of saturated to unsaturated fatty acids. Arterial essential and total amino acid (AA) concentrations in plasma and whole blood were elevated when cows were fed 41% versus 33% UIP. However, mammary arteriovenous differences, extraction percentages, and uptakes of most AA were not significantly affected by dietary treatments. Across treatments, peptide AA accounted for ~10% of AA in arterial whole blood but no net uptake of peptide AA by the lactating gland was detected. Results indicated that dietary fat supplementation at two levels of UIP can increase milk production by altering mammary lipid metabolism, thereby improving the efficiency of milk synthesis. However, depression of milk protein percentage in response to dietary fat supplementation was not alleviated by elevating arterial essential and total AA through higher dietary UIP. / Ph. D.