We demonstrate that the brain spectrin isoforms (240/235) and (240/235E) are present in all mammalian species studied (human, bovine, mouse, and rat). Immunohistochemistry with a panel of eleven polyclonal antibodies have indicated an identical localization of the brain spectrin isoforms in all mammalian species. Brain spectrin(240/235) is found primarily in axons, and brain spectrin(240/235E) primarily in cell bodies and dendrites. Immunoprecipitation and Western blotting studies have indicated that the subunit molecular weights of brain spectrin(240/235) and (240/235E) are identical in all mammalian species. We demonstrate that when proteolysis is not completely blocked during immunoprecipitation studies, the 235 kDa subunits are converted to a 230 kDa polypeptide [brain spectrin(240/235)] and a 232 kDa polypeptide [brain spectrin(240/235E)]. Finally, we show that both the alpha and beta subunits of brain spectrin(240/235) and brain spectrin(240/235E) are antigenically distinct in every species examined. These studies indicate that previous findings on the structure, location, and function of mouse brain spectrin isoforms can now be generalized to all mammalian species.