Ten multiparous and 5 primiparous cows (62±33 d in milk) were used in a replicated 5×5 Latin square design to investigate three dietary protein evaluation systems for lactating cows: milk protein score, Cornell Net Carbohydrate and Protein System, and an increase in Lys and Met to supply 15 and 5% of the predicted total essential amino acids (AA) in duodenal digesta, respectively. Five isonitrogenous diets were evaluated differently by the three systems. The milk protein score indicated that formulations of diets 2, 3, 4, and 5 were of equal quality and better for milk protein synthesis than was diet 1. The Cornell Net Carbohydrate and Protein System indicated that diets 3, 4, and 5 contained adequate AA, but diets 1 and 2 were deficient in Lys and Met. The increase in Lys and Met supply indicated that only diets 3 and 5 provided adequate Lys and Met. The dry matter intake, energy-corrected production, and fat production were lower, and 3.5% fat-corrected milk production tended to be lower, for cows consuming diets 4 and 5 than for cows consuming diets 2 and 3. Cows consuming diet 1 had lower percentages of crude protein, true protein, and casein N in their milk than did with cows consuming all other diets. Cows fed diets 3 and 5 had higher percentages of protein in milk than did cows fed other diets. Protein production was greater, and casein N tended to be greater, for cows consuming diet 3 than for cows consuming diet 2. Although all three systems had validity, the system by which Lys and Met were increased most accurately predicted milk protein percentages and production.