The effect of the supply of metabolizable protein (MP) on protein metabolism across the splanchnic tissues was determined in six catheterized lactating Holstein cows. In a crossover design, two isonitrogenous (16.3% CP) diets balanced to provide a low (Lo-MP) or high (Hi-MP) supply of MP were fed over 35-d periods. After 24 d of feeding, N balance was determined over a 6-d period. On d 33, [13C] sodium bicarbonate was infused into one jugular vein for 6 h, and hourly breath samples were collected. On d 34 or 35, L[1-(13)C] leucine was infused into one jugular vein, and between 2 to 6 h of infusion, breath and blood samples were taken hourly from the portal and hepatic veins and an artery. Isotopic enrichments of plasma leucine, 4-methyl-2-oxopentanoate, and expired CO2 were determined for calculation of leucine kinetics. Net leucine absorption was greater, either on a direct basis (leucine transfer only) or corrected for portal-drained viscera metabolism to 4-methyl-2-oxopentanoate and CO2 for the Hi-MP diet. There were no effects of diet on hepatic net flux of leucine across the liver, and, thus, more leucine was available to peripheral tissues with the Hi-MP diet. Combined with an increment in portal absorption of most of essential AA, this led to increased milk protein output, although it only represented 16% of the additional available leucine. Whole body leucine oxidation was also greater for the Hi-MP diet, as was leucine used for protein synthesis. Despite these changes, MP supply did not affect irreversible loss rate of leucine by portal-drained viscera and the liver; these averaged 35 and 20% of whole body irreversible loss rate, respectively. These ratios confirm the high metabolic activity of splanchnic tissues in lactating dairy cows, which are even greater than previously reported in growing ruminants.