The influence of amino acid nutrition on the response to milking more frequently, with or without injection of growth hormone, was examined in eight dairy cows in two 4 x 4 Latin squares with 28-d periods. The four treatments were a diet adequate in amino acids with or without injection of growth hormone and a diet inadequate in amino acids with or without injection of growth hormone. For all four treatments, during the last 14 d of each period, one half of the mammary gland was milked three times a day (3x), while the other half remained on twice-daily milking (2x). Both diets were based on grass silage given ad libitum and 4 kg/d of sugar beet pulp together with a supplement containing either fish meal (adequate diet) or feather meal (inadequate diet) as the only protein feeds. The diet containing feather meal is known to be deficient in His, Met, and Lys. On the fish meal diet, the cows responded positively to growth hormone and to milking more frequently and the responses to both treatments were additive. On the feather meal diet, however, even though injection of growth hormone increased the yield of milk protein by around 10%, milking more frequently did not affect milk production. It is concluded that milking more frequently has a weaker effect on the partitioning of amino acid use between body and udder than does growth hormone treatment.