Abstract: Unidirectional L-phenylalanine transport into six brain regions of pentobarbital-anesthetized rats was studied using the in situ brain perfusion technique. This technique allows both accurate measurements of cerebrovascular amino acid transport and complete control of perfusate amino acid composition. l-Phenylalanine influx into the brain was sodium independent and could be described by a model with a saturable and a nonsaturable component. Best-fit values for the kinetic constants in the parietal cortex equaled 6.9 × 10−4 Mmol/s/g for Vmax, 0.011 μmol/ml for Km, and 1.8 × 10−4 ml/s/g for Kd during perfusion with fluid that did not contain competing amino acids. d-Phenylala-nine competitively inhibited l-phenylalanine transport with a Ki∼ 10-fold greater than the Km for l-phenylalanine. There were no significant regional differences in Km,Kd, or Ki, whereas Vmax was significantly greater in the cortical lobes than in the other brain regions. l-Phenylalanine influx during plasma perfusion was only 30% of that predicted in the absence of competing amino acids. Competitive inhibition increased the apparent Km during plasma perfusion by ∼20-fold, to 0.21 pmol/ml. These data provide accurate new estimates of the kinetic constants that describe L-phenylalanine transport across the blood-brain barrier. In addition, they indicate that the cerebrovascular transfer site affinity (1/Km) for L-phenylalanine is three- to 12-fold greater than previously estimated in either awake or anesthetized animals.