In previous studies, the localization of a pertussis toxin-sensitive G protein was demonstrated in ependymal cilia, but the identification of the subtype of G protein was inconsistent. To clarify this issue, we studied the localization of Goα, Gi1α, Gi3α and Gi2α in the ciliated ependymal cells and in the cilia of some other tissues of rats using specific antibodies. The cilia of the ependymal cells that line the ventricular cavity of the brain were intensely immunoreactive for Gi2α, but not for Goα, Gi1α or Gi3α. Immunoblot analysis demonstrated higher levels of Gi2α in the ependymal cilia-rich pellet than in the motor area of the parietal cortex. At the ultrastructural level, the immunoreactivity specific for Gi2α was found predominantly in the cilia, but rarely in the microvilli or the basal bodies of ependymal cells. In cross-sections, the immunoreactivity specific for Gi2α was observed only in cell membranes, in particular, in the inner electron-dense leaflet of the trilaminar structure. In addition to that in the ependymal cilia, such specific localization of Gi2α was observed in the motile cilia in other tissues, including the oviduct and trachea. By contrast, the stereocilia in the ductus deferens were not immunopositive for Gi2α. These findings suggest that Gi2 might play an important role in the signal transduction in ciliary membrane-associated function(s) of the ependymal cells, oviduct and trachea.