Recent cloning of K+ channel beta subunits revealed that these cytoplasmic polypeptides can dramatically alter the kinetics of current inactivation and promote efficient glycosylation and surface expression of the channel-forming alpha subunits. Here, we examined the expression, distribution, and association of two of these beta subunits, Kv beta 1 and Kv beta 2, in adult rat brain. In situ hybridization using cRNA probes revealed that these beta-subunit genes are heterogeneously expressed, with high densities of Kv beta 1 mRNA in the striatum, CA1 subfield of the hippocampus, and cerebellar Purkinje cells, and high densities of Kv beta 2 mRNA in the cerebral cortex, cerebellum, and brainstem. Immunohistochemical staining using subunit-specific monoclonal and affinity-purified polyclonal antibodies revealed that the Kv beta 1 and Kv beta 2 polypeptides frequently co-localize and are concentrated in neuronal perikarya, dendrites, and terminal fields, and in the juxtaparanodal region of myelinated axons. Immunoblot and reciprocal co-immunoprecipitation analyses indicated that Kv beta 2 is the major beta subunit present in rat brain membranes, and that most K+ channel complexes containing Kv beta 1 also contain Kv beta 2. Taken together, these data suggest that Kv beta 2 is a component of almost all K+ channel complexes containing Kv 1 alpha subunits, and that individual channels may contain two or more biochemically and functionally distinct beta-subunit polypeptides.