The anatomical and morphological distribution of the G proteins G(o), G(i1) and 2, and Gs alpha-subunits in rat forebrain sections was determined using immunohistochemical techniques. Diffuse G(o) labeling occurred in the neuropil throughout the cortex, superficial layers of the entorhinal cortex, thalamus, several white matter fiber tracts, and hippocampus. G(i1) and 2 immunoreactivity was also located in the neuropil but produced a more fibrous pattern. Fibrous labeling of G(i1) and 2 was observed in the cortex, amygdala, hippocampal subfield CA3, and several white matter fiber tracts. Both G(o) and G(i1) and 2 labeling was present in the pencil fibers within the striatum and lateral geniculate nucleus. Gs labeling, in contrast to G(o) and G(i1) and 2, was generally cytoplasmic. Cytoplasmic Gs labeling was observed in the thalamus, habenula, dentate, geniculate nucleus, hypothalamus, and hippocampus. Intense Gs labeling was observed in the striatum parenchyma, choroid plexus, and infundibular stem. Based on our results, we conclude that the G proteins G(o), G(i1) and 2, and Gs are anatomically distributed differently throughout the brain. The diffuse neuropil labeling of G(o), fibrous neuropil labeling of G(i1) and 2, and cytoplasmic labeling of Gs strongly suggests that the G proteins are also differentially distributed morphologically within a neuron. The differential anatomical and cellular location of G proteins in the CNS may contribute to the coupling specificity between neurotransmitter receptors and G proteins.