The effect of ruminally protected Met (RPM) on splanchnic metabolism was measured in 3 primiparous and 3 multiparous Holstein cows. Doses of RPM (0, 36, and 72 g/d) were tested in a replicated 3 x 3 Latin square design, over 3 consecutive 14-d experimental periods. A mixed ration was fed in 12 equal meals per d (average dry matter intake: 17.5 +/- 0.08 kg/d). Indwelling catheters were surgically implanted in the mesenteric artery and the portal and hepatic veins for blood collection, as well as in 2 distal branches of the mesenteric vein for infusion of p-aminohippurate to determine blood flow. On d 14 of each period, a temporary catheter was inserted into a mammary vein and 6 hourly blood samples were collected to determine plasma concentrations of metabolites, hormones, and their respective fluxes across the splanchnic bed and mammary glands. Yields of milk (32.8, 32.0, and 32.9 +/- 0.92 kg/d) and protein (1,028, 1,053, and 1,075 +/- 28.7 g/d) were unaffected by level of RPM. However, the true protein content in milk from primiparous cows increased linearly (2.92, 3.09, and 3.34 +/- 0.077%). The addition of RPM linearly increased the net flux of Met across the portal-drained viscera, which resulted in increased arterial Met concentrations (25, 29, and 40 +/- 1.1 microM). Although it had no significant effect on net portal and hepatic fluxes of other essential amino acids, RPM resulted in a linear increase in the total splanchnic output of Ile, Leu, Phe, and Thr. These results suggest that feeding RPM triggered a homeostatic response resulting in less utilization of certain essential amino acids through the gastrointestinal tract and liver. Net mammary uptake of Met did not change with the addition of RPM. However, mammary extraction of Met decreased in a linear fashion in response to increased arterial inflow.