We investigated the expression of the genes encoding Rab proteins, low molecular weight GTP-binding proteins, in the rat parotid gland by the use of reverse transcription-polymerase chain reaction, and detected cDNAs of Rab3D, Rab4, and Rab26. We further examined the characteristics and localization of Rab26 by western blotting and light and electron microscopic immunocytochemistry. Western blotting using an antibody against the Rab26-specific, C-terminal peptide detected the His-tagged Rab26 protein as a single 27-kDa band. This band also displayed binding to [alpha-32P]GTP. The fraction containing secretory granule membranes in an acinar cell homogenate was immunostained with the antibody. Supporting this, the immunocytochemical reaction for Rab26 was localized immediately around secretory granules in the acinar cells. The immunostaining disappeared from the acinar cells after treatment of rats with isoproterenol. These findings suggest that Rab26 participates in the regulated secretion of granules and functionally belongs to the Rab3 group.