Lysine (Lys) utilization, excretion, and contribution to other amino acids (AA; transamination after oxidation) were explored using a stable isotope technique. Four ruminally-cannulated cows in a 4x4 Latin square (17-d periods) received Lys at 0, 25, or 50 g/d into the abomasum via continuous infusion for the last 5 d (L0, L25, or L50, respectively) or rumen-protected Lys at 105 g/d (42 g/d Lys) for 17-d. Body labile Lys pool was labeled with 15N-Lys via continuous abomasal infusion (last 5 d) during which total collection of milk, urine, and feces and blood sampling were conducted. Steady-state enrichment of 15N in samples occurred on d 4. Digestible Lys (dLys) supply was calculated by 15N absorbed (infused fecal excretion) divided by 15N enrichment of milk Lys. 15N partitioned into milk and urine was used to calculate the transfer of dLys to milk and urine. 15N enrichment of blood and milk AA was determined for Lys contribution to other AA assuming zero net Lys uptake of peripheral tissues. Data were analyzed using SAS-PROC MIXED (cow as random and treatment as fixed effects). All data from L0 were removed from analysis due to issues of abomasal infusion. Fecal 15N excretion was 12% of 15N-Lys infused across treatments, indicating free Lys in the small intestine is not 100% digestible. Increasing Lys supply from L25 to L50 increased dLys (P=0.05) from 132 to 152 g/d and increased dLys that was utilized for milk (108.1 to 117.5 g/d, P=0.02) and numerically increased that was excreted in urine (23.6 to 34.2 g/d; P=0.21). Proportion of dLys transferred to milk (80%) and urine (20%) were not affected by dLys supply. 15N enrichment occurred in a few AA in plasma (Glu and Ala), but enrichment occurred for almost all milk AA (relatively high in Glx, Asx, and branched chain AA). Enrichment in milk AA was significantly or numerically greater for L50 vs. L25. In conclusion, results confirmed large flexibility of the mammary glands to supply AA through transamination. Increasing dLys increased Lys uptake by the mammary gland but also increased Lys oxidation contributing to other AA for milk protein.