Uptake of L-2,4-diaminobutyric acid (DABA), a positively charged analogue of gamma-aminobutyric acid (GABA), by a synaptosomal fraction isolated from rat brain occurred with a Km of 54 +/- 12 microM and a Vmax of 1.3 +/- 0.2 nmol/min/mg protein. The transport of DABA was inhibited competitively by GABA whereas that of GABA was affected in the same manner by addition of DABA. The maximal accumulation of DABA ([DABA]i/[DABA]c) was observed to increase as the second power of the transmembrane electrical potential ([K+]i/[K+]e) and the first power of the sodium ion concentration gradient. These findings indicate that DABA is transported on the GABA carrier with a net charge of +2, where one charge is provided by the cotransported Na+ and the second is contributed by the amino acid itself. Since uptake of GABA, an electroneutral molecule, is accompanied by transfer of two sodium ions, the results obtained with DABA suggest that one of the sodium binding sites on the GABA transporter is in proximity to the amino acid binding site.