Brain protein synthesis was measured in anesthetized adult, male Sprague–Dawley rats by an in situ internal carotid arterial perfusion technique using [3H]leucine. The specific activity of free intracellular leucine and of tRNA leucine were determined by HPLC separation of phenylisothiocyanate (PITC) derivatives of amino acids. The specific activity of the leucyl-tRNA pool rapidly equilibrated with the free intracellular leucine pool within 2 min. The specific activity of the tRNA and free leucine pools in brain reached equilibrium by 10 min. Plasma amino acid specific activity, however, remained threefold higher than the specific activity of tRNA and free leucine pools. Estimates of protein synthesis were 0.62 ± 0.06 nmol/min/g and were constant between 10 and 30 min of perfusion. The in situ perfusion model for protein synthesis described is a controlled system suited to measurements of protein synthesis in brain that can be applied to the study of brain metabolism under changing physiological conditions.