Experimental objectives were to evaluate the effects of supplementation with rumen-protected methionine (RPM) from 31 ± 2 until 127 ± 2 DIM on circulating amino acid concentrations and lactation performance of dairy cows. Holstein cows (n = 309) were housed in a freestall barn, milked twice daily, fed a basal diet formulated to 16.7% CP to deliver 2521 g of metabolizable protein (MP) with 6.93 lysine as % of MP and randomly assigned to once daily top-dressing with either: 1) RPM, 21.2 g of Smartamine M mixed with 38.8 g of dry distillers grains (2.34 methionine as % of MP) or 2) Control (CON), 60 g of dry distillers grain (1.87 methionine as % of MP). Plasma was assayed for free amino acids by gas chromatography using a commercial kit (EZ:faast, Phenomenex). Amino acid data were analyzed using a linear mixed model with repeated measures. Milk yield and composition were determined monthly and analyzed using a linear mixed model with treatment as a fixed effect and enrollment week as a random effect. Cows treated with RPM had increased milk protein content at all 3 milk tests (mean 58.7, 86.0, 113.5 DIM) (2.96 vs. 3.03%, P = 0.003; 3.02 vs. 3.07%, P = 0.02; 3.05 vs. 3.12%, P = 0.02) with no difference in milk yield. Blood samples were collected from a subset of cows (n = 8 CON; n = 12 RPM) at 0, 3, 6, 9, 12, 18, and 24 h after feeding RPM. Plasma methionine did not differ between treatments at 0 (P = 0.37) and 3 h (P = 0.50) but was greater in RPM cows at 6 (P = 0.03) and 9 h (P < 0.0001), peaking at 12 h (52.4 vs 26.0 nmol/mL, P < 0.0001), decreasing by 18 h (P = 0.12), and back to basal by 24 h (P = 0.44). Plasma lysine (P = 0.47) and histidine (P = 0.42) were unaffected by treatment. Another subset of cows (n = 16 CON; n = 24 RPM) was evaluated at 12 h after top-dressing. Cows fed RPM had increased methionine in both primiparous (23.9 vs. 46.4; P = 0.007) and multiparous (23.5 vs. 38.7; P = 0.02) cows. Thus, top-dressing RPM resulted in a surprisingly large but acute change in circulating methionine and a small but consistent change in milk protein content.