To investigate whether splanchnic uptake of the indicator amino acid ([1-(13)C] phenylalanine) during the fed state alters the estimate of lysine requirement, five healthy men were studied at graded levels of lysine intake, with either an oral or intravenous (IV) tracer protocol, in a randomized, crossover design. Splanchnic extraction of the oral tracer was expressed as the difference between the ratio of the enrichments in urinary phenylalanine between tracer protocols. The rate of release of (13)CO(2) from (13)C-phenylalanine oxidation (F(13)CO(2)) was measured and a two-phase linear regression crossover model was applied to determine the lysine requirement. Mean splanchnic extraction of the oral tracer was approximately 19%. Although actual F(13)CO(2) was higher during oral tracer infusion (P < 0.001), the breakpoint was not different from that determined with IV infusion (P = 0.98), with both yielding a mean lysine requirement of 36.6 mg/(kg. d). The upper 95% confidence intervals were 52.5 and 53.3 mg/(kg. d) for the oral and IV isotope infusions, respectively. These results demonstrate that routes of isotope administration using the indicator amino acid oxidation technique do not affect the estimated amino acid requirement. Therefore, the indicator amino acid oxidation method using the oral route, which is less invasive and allows for studies in vulnerable groups such as infants and children, should be the preferred method for studying amino acid requirements.