While the γ-aminobutyric acid (GABA) transporter GAT1 exclusively transports GABA, GAT2, -3, and -4 also transport β-alanine. Cross-mutations in the external loops IV, V, and VI among the various GABA transporters were performed by site-directed mutagenesis. The affinity of GABA transport as well as inhibitor sensitivity of the modified transporters was analyzed. Kinetic analysis revealed that a cross-mutation in which loop IV of GAT1 was modified to resemble GAT4 resulted in increased affinity to GABA from Km = 8.7 to 2.0 μM without changing the Vmax. A cross-mutation in loop VI, which swapped the amino acid sequence of GAT2 for GAT1, decreased the affinity to GABA (Km, 35 μM). These results suggest that loops IV and VI contribute to the binding affinity of GABA transporters. A substitution of three amino acids in loop V of GAT1 by the corresponding sequence of GAT3 resulted in β-alanine sensitivity of its GABA uptake activity. These three amino acids in loop V seem to participate in the β-alanine binding domain of GAT3. It is suggested that those three external loops (IV, V, and VI) form a pocket in which the substrate binds to the GABA transporters.