We describe a method based on the traditional arterio-venous balance technique in conjunction with muscle biopsies for the determination of leg muscle protein balance during the non-steady state in blood amino acid concentrations. Six young, healthy individuals were studied in the postabsorptive state (pre-Phe) and following a bolus ingestion of ~ 0.5 g phenylalanine (post-Phe). Post-Phe free phenylalanine concentrations in blood and muscle increased (P < 0.05), but the respective concentrations of the amino acid threonine did not change. The average post-Phe leg net balance (NB) for threonine decreased from basal (P < 0.05), but that for phenylalanine did not change. A volume of distribution for free phenylalanine in the leg was calculated based on the leg lean mass and the relative muscle water content and used to estimate the rate of accumulation of free phenylalanine in the leg. When the post-Phe NB for phenylalanine was corrected for the rate of accumulation of free phenylalanine in the leg, the post-Phe NB for phenylalanine decreased from basal (P < 0.05). This corrected value was not different (P > 0.05) from the value predicted for the phenylalanine NB based on the pre- and post-Phe NB responses for threonine. We conclude that the protein net balance in non-steady state blood phenylalanine concentrations can be determined from the arterio-venous phenylalanine NB by accounting for changes in free phenylalanine within its volume of distribution.