Two genes encode two forms of glutamate decarboxylase, GAD65 and GAD67. Because the two GADs differ in subcellular distribution and interactions with the cofactor pyridoxal phosphate, the two enzymes may play different roles in gamma-aminobutyric acid (GABA) production. In this study we have used in situ hybridization to compare the regional and cellular distributions of the two GAD mRNAs in rat brain. Both GAD mRNAs are abundant in olfactory bulb, olfactory tubercle, zona incerta, reticular nucleus of the thalamus, oculomotor nuclei, and pontine tegmental area. GAD65 mRNA is more abundant in several structures of the visual system, including the lateral geniculate nuclei, superior colliculi, and olivary pretectal nucleus, as well as in several hypothalamic and pontine nuclei. In contrast, GAD67 mRNA is more abundant in neocortex, the granular layer of olfactory bulb, lateral and medial septum, globus pallidus, inferior colliculi, and cerebellar cortex. Both GAD mRNAs are present in interneurons as well as in projection neurons, and both are present in neurons with different types of synapses, including dendrodendiritic, axosomatic, and axodendritic synapses. GAD65 mRNA predominates in the visual and the neuroendocrine systems, which are more subject to phasic changes, while GAD67 is present at relatively higher concentrations in many tonically active neurons. GAD65 and GAD67 together may provide more flexibility in the regulation of GABA synthesis than either could alone. © 1993 Wiley-Liss, Inc.