The objective of this study was to quantify the utilization of dietary and systemic phenylalanine for mucosal and hepatic constitutive protein synthesis in piglets. Seven female piglets (7.6 kg) bearing arterial, portal, peripheral venous, and gastric catheters were fed a high-protein diet and infused intragastrically with U-13C-labeled protein and intravenously with [2H(phenyl)5]phenylalanine ([2H5]phenylalanine) for 6 h. The isotopic enrichment of the two phenylalanine tracers was measured in arterial and portal blood, in mucosal and hepatic-free and protein-bound phenylalanine, and in very low-density apolipoprotein B-100, albumin, and fibrinogen. The relative isotopic enrichments of the tracers in mucosal-free (ratio of 2H5- to U-13C-labeled = 0.20 +/- 0.05) and protein-bound (0.32 +/- 0.08) phenylalanine differed significantly (P < 0.01). Although this suggests preferential use of arterial phenylalanine for mucosal protein synthesis, on a molar basis, 59 +/- 6% of the mucosal protein was derived from dietary phenylalanine. There were significant differences (P < 0.025) between the relative labeling of the two tracers in arterial (ratio of 2H5- to U-13C-labeled = 1.25 +/- 0.48) and portal (ratio of 2H5- to U-13C-labeled = 0.72 +/- 0.18) phenylalanine. The mean ratio of the two tracers in all proteins of hepatic origin that were analyzed (0.69 +/- 0.18) was similar to that of portal phenylalanine. We conclude that in the fed state portal phenylalanine is preferentially used for constitutive as well as secreted hepatic protein synthesis.