In vitro labeling of guanine nucleotide binding proteins has been accomplished for the microscopic localization and quantitation of these sites in individual brain regions. Cryostat sections of rat brain were labeled with [3H]guanylyl-5'-imidodiphosphate [( 3H]Gpp(NH)p) using parameters which resulted in a high specific to non-specific (signal to noise) binding ratio. Gpp(NH)p binding sites were identified to some extent in all brain regions examined. The highest densities existed in the olfactory bulb, cerebral cortex, caudate-putamen, interpeduncular nucleus, substantia nigra and cerebellum. Thus, GTP binding proteins can be labeled and localized using a novel application of an autoradiographic technique developed for microscopic detection of receptors.