Glutamic acid decarboxylase (GAD) catalyzes synthesis of the inhibitory neurotransmitter gamma-amino butyric acid. Two homologous forms of GAD encoded by separate genes have been cloned from rat brain, with predicted protein sizes of 67 and 65 kilodaltons. GAD is present outside the brain, and pancreatic islet GAD is believed to be a target of autoimmunity in insulin-dependent diabetes mellitus. However, peripheral expression of the two GAD genes is incompletely characterized. We, therefore, investigated GAD expression in peripheral tissues, including pancreas, of mouse and rat. cDNAs encoding GAD 67 and GAD 65 were cloned from mouse brain and shown to be 95% homologous with the rat sequences. RNase protection assay using specific cRNA probes demonstrated expression of both GAD forms in freshly harvested pancreas and testis. Levels of both GAD mRNAs were greater in rat than mouse pancreas. GAD 67 mRNA was more abundant than GAD 65, and both were localized to islet beta-cells by in situ hybridization. In testis, both GAD mRNAs were localized to spermatocytes. Additionally, GAD 67, but not GAD 65, mRNA was detected in mouse and rat spleen and mouse liver. Thus, both GAD genes are expressed in peripheral tissues, with GAD 67 mRNA being more abundant under physiological conditions. The expression of both GAD 67 and GAD 65 genes specifically in islet beta-cells indicates that both GAD forms are candidate autoantigens in rodent models of insulin-dependent diabetes mellitus.