The regional distributions of the G protein beta subunits (G beta 1-beta 5) and of the G gamma 3 subunit were examined by immunohistochemical methods in the adult rat brain, In general, the G beta and G gamma 3 subunits were widely distributed throughout the brain, with most regions containing several G beta subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense G beta immunostaining. Negative immunostaining was observed in cortical layer I for G beta 1 and layer IV for G beta 4, The hippocampal dentate granular and CA1 - CA3 pyramidal cells displayed little or no positive immunostaining for G beta 2 or G beta 4. No anti-G beta 4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for G beta 1 was absent from the cerebellar molecular layer, and G beta 2 was not detected in the Purkinje cells. No positive G gamma 3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells, Double-fluorescence immunostaining with anti-G gamma 3 antibody and individual anti-G beta 1-beta 5 antibodies displayed regional selectivity with G beta 1 (cortical layers V-VI) and G beta 2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of G beta 1-beta 5 with G gamma 3, specific dimeric associations in situ were observed within discrete brain regions.