Although in dairy cows the mammary gland (MG) is the major net user of essential AA (EAA) supply, milk protein synthesis from absorbed EAA is not a straightforward process. Early studies identified 2 groups of EAA based on different pattern of mammary utilization: group 1 [Met, Phe (+Tyr), Trp], where MG uptake was similar to secretion in milk protein, and group 2 (Arg, Ile, Leu, Lys, Thr, and Val), where uptake exceeded milk protein output. This review examines the validity of this classification under variable protein supply through a meta-analysis, with the outcomes then explained with studies in which the fates of individual EAA were monitored using isotope approaches. For the meta-analysis, the Fick principle, based on stoichiometric transfer of Phe+Tyr uptake to milk protein, was used to estimate mammary plasma flow across all studies. This approach was judged acceptable because doubling Phe supply did not result in mammary oxidation of Phe+Tyr and either limited or no contribution of peptides to Phe and Tyr mammary supply could be detected. The AA content of proteins synthesized by the MG was estimated from milk protein composition, and the uptake-to-output ratio (U:O) for individual AA was re-calculated based on these assumptions. Analysis of individual samples by isotopic dilution resulted in reduced variance compared with analysis on pooled samples performed with an AA analyzer. Globally, the U:O of His and Met is maintained close to unity under variable protein supply. The group 2 AA could be subdivided. First, the U:O for group “2v” AA (Ile, Leu, Val, and Lys) is greater than 1 and varied with protein supply. Accordingly, the increased U:O of Leu, induced by duodenal casein infusion, led to extra-mammary Leu oxidation. Decreasing Lys supply decreased Lys U:O and the associated transfer of N to non-EAA, mainly to Glx, Asx, Ser, and Ala. Second, the U:O of group “2nv” AA, Arg and Thr, does not vary with protein supply. The Arg U:O averages 2.5, whereas the Thr U:O, albeit averaging 1.2, does not differ from unity. Excess of both these AA is probably directed toward the synthesis of non-EAA rather than energy supply. Overall, the ability of the MG to use excess EAA-N supply offers alternative sources of N and C for energy provision, lactose synthesis and non-EAA synthesis. The latter function spares dietary non-EAA for other necessary processes, such as gluconeogenesis and energy supply, in other tissues to support lactation.