AbstractMilk production responses to protein supplementation of grass silage diets were investigated in two experiments with dairy cows. In Experiment 1. four cows received a basal diet of grass silage ad libitum plus a barley/soya supplement at 5 kg day−1. The cows received the following treatments in a 4 × 4 Latin square design with 10‐day periods: (1) basal diet, (2) basal diet plus 230 g casein day−1 infused into the abomasum; (3) basal diet plus 225 g soya protein isolate (SPI) day−1 into the abomasum; and (4) basal diet plus a dietary addition of 1.2 kg fishmeal‐based product (AP) day−1. All three supplements increased (P < 0.05) the yield of milk and milk protein relative to the basal diet, the values being 16.9. 535:18.8. 610; 19.5, 625; and 18.5 kg day−1 and 582 g day−1 for the basal. AP, casein and SPI treatments respectively. Responses to casein were greater (P < 0.05) than to SPI. Changes in the concentrations of amino acids in blood plasma suggested that methionine, tryptophan and phenylalanine were in shortest supply relative to demand, and their potential role as limiting amino acids was investigated in a second experiment.In Experiment 2, four dairy cows were used in a 4 × 4 Latin square design with 10‐day periods. The four treatments were (1) a diet of grass silage ad libitum plus 5 kg day−1 of a barley/soya supplement containing 176 g CP kg−1 DM (LP); (2) LP plus an intravenous infusion of 5.0 g methionine, 9.1 g phenylalanine and 2.2 g tryptophan day−1; (3) grass silage ad libitum plus 5 kg day−1 of a barley/soya supplement containing 334 g CP kg−1 DM (HP); and (4) HP plus the intravenous infusion of methionine, phenylalanine and tryptophan. The HP treatment produced a higher yield (P < 0.05) of milk protein than the LP treatment (722 vs. 667 g day−1) but the amino acid infusion had no effect with either treatment.The elimination of methionine, phenylalanine and tryptophan (and, in previous experiments, methionine and lysine) as candidates for limiting amino acids on diets of grass silage plus soya‐based supplements questions whether the differences in responses to casein and SPI can be explained in terms of their amino acid compositions. The role of factors such as peplides, producing during digestion, some of which may be biologically active, deserves investigation.