Clinicians and researchers studying protein metabolism in vivo , typically use isotopically-labeled free amino acids as metabolic tracers rather than isotopically-labeled proteins because such proteins are commercially unavailable. However, the use of free amino acids in lieu of protein tracers violates the critical assumption that tracer molecules undergo the identical biochemical reactions as the tracee molecules of interest. To address this problem we synthesized 13 C-labeled proteins using egg laying hens and investigated the relationship between tracer dose and method of delivery on 13 C-protein production. We enriched hens with one of two isotope tracers ( 13 C-1-leucine or a uniformly labeled 13 C-amino acid mixture) mixed in their food or dissolved in their drinking water at different dosing levels (86-432 mg day -1 ). The recovery of 13 C in egg white proteins of the hen fed 13 C-leucine ranged from 14% to 21%; recovery rates were highest at the lowest dosing level. At the highest dosing level egg whites were enriched more than 150‰ above background levels of 13 C. The time required for half maximal 13 C enrichment depended chiefly on the mode of tracer administration, and ranged from 2.5 days for 13 C-leucine dissolved in water to 4.9 days for 13 C-leucine mixed in food. Relative rates of 13 C recovery in the egg protein were lowest for hens fed the uniformly 13 C labeled amino acid mixture, presumably because of the high proportion of nonessential amino acids. The time required for the 13 C-enrichment in eggs to return to background levels at the end of the enrichment period was about twice that required to initially reach isotopic equilibrium with the diet, indicating significant biochemical discrimination of endogenous 13 C amino acids. We conclude that delivering small amounts of 13 C amino acid tracers in the drinking water of hens is the most effective way to produce 13 C-enriched proteins to for tracer studies that do not require delt 13 C-enrichment above 200‰.