We examined leucine and lysine metabolism in adult humans (n = 8 and n = 5, respectively) to assess the current estimate of protein requirement. Each subject consumed a controlled isoenergetic liquid diet for three 10-d periods at three intakes of protein, 0.6, 0.8 and 1.0 g.kg-1.d-1, in random order. Measurements of flux and catabolism were determined in each subject in both the fed and postabsorptive states, with a primed, 4-h continuous infusion of L-[1-13C]leucine and L-[alpha-15N]lysine. Fluxes of leucine and lysine were not affected by feeding state or protein intake. Adaptation to different protein intakes did not affect the oxidation of leucine and lysine in the postabsorptive state, but leucine oxidation was significantly higher during the fed period compared with the postabsorptive period at a protein intake of 1.0 g.kg-1.d-1 (P less than 0.05) and tended to be greater (P = 0.06) at an intake of 0.8 g.kg-1.d-1. Nitrogen balance assessed over the last 72 h of each dietary period was positive for all subjects at protein intakes of 0.8 and 1.0 g.kg-1.d-1 of protein and, hence, was consistent with the leucine oxidation data. These data suggest that experiments conducted during the postabsorptive period are not appropriate for estimating protein requirements. Leucine oxidation data obtained during the fed state estimate a protein requirement for young men of between 0.6 and 0.8 g.kg-1.d-1.