Mammary gland protein metabolism, determined by an arteriovenous difference technique, was monitored in four Holstein-Friesian dairy cows in response to supplemental dietary protein (provided as rumen-protected soyabean meal) during late lactation (weeks 24–30). Each cow was offered two isoenergetic diets composed of grass silage (170 g crude protein/kg dry matter) plus either a low (108 g/kg) or medium (151 g/kg) crude protein concentrate in a single crossover design involving two 21 d periods. On day 21, arteriovenous measurements across the mammary gland were made during a 13 h continuous i.v. infusion of [1-13C]leucine and with frequent (2 hourly) milk sampling during the final 6 h. Although total milk yield was slightly increased (+1 kg/d) by protein supplementation, milk protein yield was not significantly affected. Whole body protein flux (protein synthesis plus oxidation) was not significantly affected by supplementation. Total mammary gland protein synthesis (milk plus non-milk protein) was also not affected by supplementation but on both diets gland synthesis was always greater (by 20–59%) than milk protein output. The fractional oxidation rate of leucine by the mammary gland was significantly increased by protein supplementation (0·047 v. 0·136). Although the enrichment of leucine in secreted milk protein continued to increase, the final value (at 13 h) was 0·94 of the arterial plasma free leucine plateau value (not significantly different), suggesting almost exclusive use of plasma free leucine for milk protein synthesis. Based on current feeding schemes for dairy cattle, a fixed proportion (0·65–0·75) of the additional protein intake (+490 g/d) should have been partitioned into milk protein. Instead, leucine oxidation by the mammary gland was increased. Whether oxidation of other amino acids was also enhanced is unknown but if amino acid oxidation and the ‘additional’ non-milk protein synthesis occurring in the gland are not crucial to milk synthesis, then by reducing such activities improvements in the efficiency of converting absorbed amino acid into milk protein can be achieved.