We have proposed a leucine requirement of 40 mg/(kg. d) in adults, based on 24-h direct amino acid balance (24-h DAAB) studies in which leucine intake is calculated as the sum of diet and tracer intake. However, it is possible that the tracer intake that is given during the fasting state in the 24-h studies is oxidized, thereby not contributing to the effective daily leucine intake and thus lowering the intake and, consequently, the requirement estimate. We assessed the fasting state leucine oxidation with different leucine infusion rates ( approximately 2.5-5% of the leucine flux rate) in well-nourished Indian men. Healthy subjects (n = 10) in a fasting state were studied during three randomly administered infusions of different, known amounts of leucine, supplying 4.1, 6.6 or 8.3 mg/(kg. 12 h) during the 12-h fast. Mean 12-h leucine oxidation rate and leucine flux for the different levels of leucine infused did not change significantly (P > 0.1) for the three leucine infusion rates. The plasma leucine concentrations increased significantly after 12 h of leucine infusion, rising from between 20 and 50 micromol/L by the end of the infusions over the range of tracer input. We conclude that tracer leucine infused in the fasting state does not measurably increase leucine oxidation at the doses studied. Thus, tracer intake during the 12-h fast contributes to the effective leucine intake in 24-h DAAB studies.