Enteral l-glutamate is extensively utilized as an oxidative fuel by the gut mucosa in the neonate. To identify major uptake pathways and to understand uptake regulation, we examined transport kinetics and molecular identities of apical membrane l-glutamate transporters in epithelial cells sequentially isolated along the small intestinal crypt-villus axis from milk protein-fed, 16-day-old pigs. The distended intestinal sac method was used to isolate 12 sequential cell fractions from the tip villus to the bottom crypt. Initial rates and kinetics of l-glutamate uptake were measured with l-[G-³H]glutamate by fast filtration in apical membrane vesicles prepared by Mg²⁺precipitation and differential centrifugation, with membrane potential clamped by SCN⁻. Initial l-glutamate uptake results suggested the presence of B^oand X_AG⁻transport systems, but the X_AG⁻system was predominant for uptake across the apical membrane. Kinetic data suggested that l-glutamate uptake through the X_AG⁻system was associated with higher maximal transport activity but lower transporter affinity in crypt than in villus cells. Molecular identity of the X_AG⁻glutamate transporter, based on immunoblot and RT-PCR analysis, was primarily the defined excitatory amino acid carrier (EAAC)-1. EAAC-1 expression was increased with cell differentiation and regulated at transcription and translation levels from crypt to upper villus cells. In conclusion, efficiency and capacity of luminal l-glutamate uptake across the apical membrane are regulated by changing expression of the X_AG⁻system transporter gene EAAC-1 at transcription and translation levels as well as maximal uptake activity and transporter affinity along the intestinal crypt-villus axis in the neonate.