Four multiparous Holstein cows with ruminal and duodenal cannulas were assigned to 4 × 4 Latin squares at peak (wk 4), early (wk 14 to 16), mid (wk 21 to 23), and late (wk 29 to 31) lactation to determine, in the presence of supplemental Met, the extent of Lys limitation and its required contribution to total essential AA in duodenal digesta. Treatments were duodenal infusions of 1) water alone or water with 2) 10 g/d of DL-Met plus 10 g/d of L-Lys, 3) 10 g/d of Met plus 20 g/d of Lys, and 4) 10 g/d of Met plus 30 g/d of Lys; quantities were reduced by 20% in late lactation. Rations were corn-based (corn and grass-legume silages, corn meal, wheat middlings, soybean meal, and distillers dried grains with solubles) and most limiting in Lys and Met. Intakes of ruminally degraded and undegraded intake protein (percentage of NRC requirements) were (peak) 115, 97; (early) 112, 83; (mid) 113, 87; and (late) 127, 96. Contribution of Lys to passage of total essential AA to the duodenum without infusions were 13.2, 12.4, 13.8, and 14.8% at the four respective stages of lactation. Extent of Lys limitation determined from responses in content and yield of milk protein approximated 25, 20, and 10 g/d during peak, early, and midlactation. Using measurements of AA flow to the duodenum and assumed intestinal availabilities of 100% for infusate Lys and 80% for microbial and ruminally undegraded intake protein AA, calculated required contributions of Lys to total essential AA passing to the duodenum were 15.2, 13.9, and 14.5% for peak, early, and midlactation, respectively.