Administration of labeled, free amino acids does not allow direct assessment of in vivo dietary protein digestion and absorption kinetics. Consequently, dietary protein sources with labeled amino acids incorporated within their protein matrix are required. The aim of the present study was to produce intrinsically l-[1-13C]phenylalanine-labeled milk and meat protein that would permit in vivo assessment of postprandial protein digestion and absorption kinetics in humans. One lactating dairy cow was continuously infused with 420 µmol of l-[1-13C]phenylalanine/min for 96 h, with plasma and milk being collected before, during, and after isotope infusion. Twenty-four hours after infusion, the cow was slaughtered to produce intrinsically labeled meat. Levels of l-[1-13C]phenylalanine enrichment as high as 40 mole percent excess (MPE) in milk and 1.5 MPE in meat protein were achieved. In a subsequent human proof-of-principle experiment, 2 healthy young males (25 ± 1 yr; 66.2 ± 5.2 kg) each ingested 135 g of l-[1-13C]phenylalanine intrinsically labeled minced beef, after which plasma samples were collected at regular time intervals. Plasma l-[1-13C]phenylalanine enrichments increased during the first 90 min following beef ingestion, reaching peak plasma enrichment levels of 0.61 ± 0.04 MPE. Whole-body net protein balance, assessed by continuous infusion of l-[ring-2H5]phenylalanine and l-[ring-2H2]tyrosine, was higher in the postprandial period compared with basal values (6.4 ± 0.1 vs. -4.5 ± 0.1 µmol/kg per h). In conclusion, the production of intrinsically l-[1-13C]phenylalanine-labeled milk and meat protein is feasible and provides functional tools to investigate in vivo protein digestion and absorption kinetics in humans.