Objectives: The proton-coupled amino acid transporter 1, hPAT1 is an absorptive transporter for amino acids such as L-Pro, Gly and g-aminobutyric acid (GABA). hPAT1 is situated in the apical membrane of intestinal epithelial cells. Little is however known about the basolateral exit pathway of amino acids that are transported into the intestinal cell via hPAT1. The aim of the present study was thus to investigate basolateral GABA transport in Caco-2 cells. Methods: Basolateral GABA transport was measured using [3H]GABA uptake in the intestinal cell line Caco-2 grown on permeable filters. The uptake was measured under different conditions: a) in the presence of 30 mM of different amino acids b) in the presence or absence of extracellular sodium or chloride c) as a function of extracellular basolateral pH d) as a function of extracellular GABA concentrations. Results: The basolateral uptake of 13 nM GABA was inhibited to approximately 50 % by amino acid such as 30 mM of Lys, Pro and Gly, whereas 30 mM mannitol had no effect on the uptake. 30 mM of b-Ala, 5-aminovaleric acid, Tau and d- aminolevulinic acid inhibited the basolateral GABA uptake to 7±0, 11±1, 18±0.2, and 25±2 % of the control uptake, respectively (N=3, average±SD). The uptake of GABA at pH 7.4 and 5.5 was both sodium and chloride dependent, and furthermore, the basolateral uptake of GABA was pH dependent. The uptake of GABA was mediated by two transporters, a high- and a low-affinity transporter, characterized by Km values of 290±180 mM and 64±14 mM, respectively. Conclusion: The results obtained in the present study identify two different transporters for GABA in the basolateral membrane of Caco-2 cells. The high-affinity transporter is likely to be sodium- and chloride dependent, whereas the low-affinity transporter is proton- dependent. There is an overlap in substrates between the basolateral transporters and hPAT1, but also notable differences in substrate specificities.