Knowledge of the amino acid sequence of a pyruvate-dependent GABA transaminase (GABA-T) from 'Arabidopsis' enabled the identification of three pyruvate-dependent isoforms in tomato. This study aimed to test the hypothesis that the three isoforms, designated as 'Le'GABA-T1, 2 and 3, are located in different subcellular compartments. Each of the four GABA-T cDNAs was fused to either the green fluorescent protein (GFP) or the myc epitope and then coexpressed with mitochondrial ([beta]ATPase) or plastidial (RecA) marker proteins in tobacco BY-2 suspension cells using particle bombardment. Analysis of the transient expression of the recombinant proteins using epifluorescence microscopy revealed that 'At'GABA-T and 'Le'GABA-T1 were localized exclusively to the mitochondrion, whereas 'Le'GABA-T2 and 'Le'GABA-T3 were localized to the cytosol and chloroplast, respectively. 'At'GABA-T, 'Le'GABA-T1 and ' Le'GABA-T3 contained N-terminal targeting sequences that were both sufficient and necessary for proper targeting. Together, these data provide evidence that multiple subcellular compartments are involved in GABA metabolism.