The existence of two distinct genes encoding two isoforms of glutamic acid decarboxylase (GAD65 and GAD67) has been demonstrated in most vertebrate classes, yet little is known about their differential distributions and functions in the central nervous system in nonmammalian vertebrates. In the present study, we have partially sequenced the cDNAs encoding GAD65 and GAD67 in the lungfish Protopterus annectens and determined their relative distributions in the adult brain by in situ hybridization histochemistry. The expression patterns of the GAD65 and GAD67 mRNAs were globally similar; the highest expression levels being observed in the granular layer of the olfactory bulb, the pallium, the subpallium, the anterior preoptic area, the thalamus, the hindbrain central gray, and the rhombencephalic visceral areas. However, striking differential expression was noticed in several structures. Very high to high concentrations of GAD67 mRNA were seen in the dorsal and ventral aspects of the anterior olfactory nucleus, which is in marked contrast to the very low expression of GAD65 in this region. Similarly, high levels of GAD67 mRNA were observed in the intermediate and ventral parts of the medial pallium that were virtually devoid of GAD65 mRNA. In contrast, GAD65 mRNA was found in the periaqueductal gray that did not express GAD67 mRNA. The differential expression of GAD65 and GAD67 mRNAs in these regions of the lungfish CNS indicates that the two GAD isoforms can be differentially regulated and that they may have distinct physiological roles.