Oral or intravenous administration of labeled, free amino acids does not allow the direct assessment of protein digestion and absorption kinetics following dietary protein intake. Consequently, dietary protein sources with labeled amino acids incorporated within the protein are required. The aim of this study was to produce milk proteins intrinsically labeled with l-[1-(13)C]phenylalanine that would allow the assessment of protein digestion and absorption kinetics and the subsequent muscle protein synthetic response to dietary protein intake in vivo in humans. Two Holstein cows (body weight of 726 +/- 38 kg) were continuously infused with l-[1-(13)C]phenylalanine at 402 micromol/min for 44 to 48 h, during and after which plasma samples and milk were collected. After milk protein separation, casein was used in a subsequent human proof-of-principle experiment. Two healthy males (aged 61 +/- 1 yr; body mass index of 22.4 +/- 0.1 kg/m(2)) ingested 35 g of casein highly enriched with [1-(13)C] phenylalanine. Plasma samples were collected at regular intervals, and skeletal muscle biopsies were collected before and 6 h after casein ingestion. In the initial experiment, a total of 5.83 kg of l-[1-(13)C]phenylalanine-enriched milk protein (casein enrichment was 29.4 molar percent excess) was collected during stable isotope infusion in the cows. In the proof-of-principle study, ingestion of 35 g of intrinsically labeled casein resulted in peak plasma l-[1-(13)C]phenylalanine enrichments within 90 min after protein ingestion (9.75 +/- 1.47 molar percent excess). Skeletal muscle protein synthesis rates calculated over the entire 6-h period averaged 0.058 +/- 0.012%/h. The production of intrinsically labeled milk protein is feasible and provides dietary protein that can be used to investigate protein digestion and absorption and the subsequent muscle protein synthetic response in vivo in humans.