The gap junction in vertebrates is encoded by the connexin gene family. Recently, a new gene family termed pannexin (Panx) has been identified in vertebrates and found to encode gap junctional proteins as well. To date, three pannexin isoforms (Panx1, 2, and 3) have been cloned from mouse and human genomes. In this study, expression of pannexins in the mouse and rat cochlea was investigated. Polymerase chain reaction and Western blot analysis showed that all three pannexin isoforms were expressed in the cochlea. Immunofluorescent staining showed that Panx1 expression was extensive. In the organ of Corti, Panx1 labeling was found in supporting cells, including pillar cells, Hensen cells, Claudius cells, and Boettcher cells. Both surface plaque-like punctate labeling and diffuse-cytoplasmic labeling were visible. However, the labeling was weak and rare in Deiters cells. No labeling was found in the hair cells. Intense labeling for Panx1 was also observed in the interdental cells in the spiral limbus, the inner and outer sulcus cells, and the type II fibrocytes in the spiral prominence and central region in the cochlear lateral wall. In addition, Panx1 labeling was detectable in Reissner's membrane and strial blood vessel cells. Panx2 labeling was restricted to the basal cells in the stria vascularis and was also detectable in the spiral ganglion neurons. However, no overlapping labeling for Panx1 and Panx2 was observed. Finally, Panx3 labeling was exclusively observed in the cochlear bone. Thus, Panx1, 2, and 3 are abundantly expressed in the mammalian cochlea and demonstrate distinct cellular distributions. Like connexins, they may play an important role in hearing.