This study examined the subcellular localization of the enzymes involved in the metabolism of 4-aminobutyrate or gamma-aminobutyrate (GABA) and the transport of this compound across membranes of higher plant cells. The subcellular localization of GABA shunt enzymes was investigated in protoplasts prepared from developing soybean cotyledons ('Glycine max' (L.) Merrill cv. Maple Arrow). Protoplast lysate was fractionated by differential and continuous Percoll-gradient centrifugation to separate organelle fractions. Glutamate decarboxylase (EC 4.1.1.15) was found exclusively in the cytosol, whereas GABA:pyruvate transaminase (EC 2.6.1.19) and succinic semialdehyde dehydrogenase (EC 1.2.1.16) were exclusively associated with the mitochondrial fractions. Mitochondrial fractions also catabolized [U-14C]GABA to labeled succinate. These result provide conclusive evidence that GABA synthesized in the cytosol is transported across the mitochondrial membranes into the matrix. In an effort to identify proteins capable of transporting GABA, heterologous complementation and characterization of known amino acid transporters were performed using a GABA-transport-deficient 'Saccharomyces cerevisiae ' mutant. AAP3 and ProT2 from 'Arabidopsis thaliana' (L.) Heynh (Landsberg 'erecta' ecotype) were identified as H+-coupled GABA transporters with Michaelis constants of 12.9 ± 1.7 and 1.7 ± 0.3 mM, respectively. The effect of external pH on the simultaneous transport of [1-14C]GABA and [2, 3- 3H]proline into yeast expressing 'ProT2' provided evidence that the charged state of GABA is an important parameter in substrate transport. ProT2-mediated [1-14C]GABA transport was inhibited by proline, choline and quaternary ammonium compounds (QACs) such as glycine betaine. Direct evidence was provided for the transport of [methyl-14C]choline. Although different metabolic routes are involved in the syntheses of these stress-related compounds, ProT2 may be a common carrier in plants for these metabolites.