The two isoforms of glutamic acid decarboxylase (GAD), GAD67 and GAD65, synthesize the neurotransmitter γ-aminobutyric acid in neurons and pancreatic β-cells. Previous studies suggest that GAD67 is a soluble cytosolic protein, whereas GAD65 is membrane-associated. Here we study the intracellular distribution of GAD67 in neurons, pancreatic β-cells, and fibroblasts transfected either with GAD65 and GAD67 together or with GAD67 alone. Neuronal GAD67 is partially recovered with GAD65 in membrane- containing pellet fractions and Triton X-114 detergent phases. The two proteins coimmunoprecipitate from extracts of brain and GAD65-GAD67 cotransfected fibroblasts, but not when extracts of GAD65 and GAD67 transfected fibroblasts were mixed and used as a stating material for immunoprecipitation. GAD67 is concentrated in the Gorgi complex region in GAD65-GAD67 cotransfected fibroblasts, but not in fibroblasts transfected with GAD67 alone. A pool of neuronal GAD67 colocalizes with GAD65 in the Golgi complex region and in many synapses. The two proteins also colocalize in the perinuclear region of some pancreatic ̄-cells. GAD67 interacts with the NH2-terminal region of GAD65, even in the absence of palmitoylation of this region of GAD65. Taken together, our results indicate that GAD65-GAD67 association occurs in vivo and is required for the targeting of GAD67 to membranes.