Connexin 31 (Cx31) mutations cause an autosomal dominant form of high-frequency hearing loss. The immunohistochemical localization of Cx31 in mouse cochlea was studied at different ages between 0 and 60 days after birth (DAB). Cx31-like immunoreactivity was detected in fibrocytes of spiral ligament and spiral limbus at 12 DAB, gradually enhanced with the increase of age and reached the adult pattern on 60 DAB. Immunoreactivity decreased gradually from the basal to apical turn in all developmental stages. The mRNA of Cx31 was also identified by RT-PCR. The distribution of Cx31 and connexin 26 were obviously different in the developing mouse cochlea. The expression and distribution of Cx31 in the development may explain the progressive hearing loss in human Cx31 mutations.