L-Glutamate is the immediate precursor of the inhibitory transmitter GABA, and considered to be supplied from alpha-ketoglutarate through a transamination reaction or from glutamine through a glutaminase reaction. In the present study, the localization of aspartate aminotransferase and glutaminase in GABAergic neurons was investigated in the rat neocortex by a double immunofluorescence method. Immunoreactivities for both soluble and mitochondrial aspartate aminotransferases were detected in more than 90% of GABA-positive neurons, whereas glutaminase immunoreactivity was not found in GABA-positive neurons. All neocortical neurons with soluble aspartate aminotransferase immunoreactivity were immunopositive for GABA, but none for glutaminase. Neurons with mitochondrial aspartate aminotransferase immunoreactivity showed either glutaminase or GABA immunoreactivity. Under confocal laser scan microscopy, immunoreactivity for soluble aspartate aminotransferase was observed in many axons and axon terminals showing immunoreactivity for glutamic acid decarboxylase, whereas immunoreactivity for mitochondrial aspartate aminotransferase was seen in only a few axons displaying immunoreactivity for glutamic acid decarboxylase. The present results indicate that soluble aspartate aminotransferase is selectively localized to cell bodies and axon terminals of GABAergic non-pyramidal neurons in the cerebral neocortex. This suggests that glutamate is supplied from alpha-ketoglutarate via transamination and works as the immediate precursor for GABA in axon terminals of GABAergic neurons. The absence of glutaminase immunoreactivity in GABAergic neurons indicates that glutamine is a "metabolically remote" precursor for GABA. Mitochondrial aspartate aminotransferase was located in perikarya, rather than in axon terminals of GABAergic neurons, suggesting a transmitter-irrelevant role of this enzyme in neurons.