Recently, we reported that homozygous males and females of a mouse model of DFN3 non-syndromic deafness generated by the deletion of Brn-4 transcription factor showed profound deafness due to severe alterations in the cochlear spiral ligament fibrocytes from the age of 11 weeks, whereas no hearing loss was recognized in young female heterozygotes. It is known that a part of obligate female carriers of DFN3 showed progressive hearing loss. In the present study, we examined the late-onset effect of Brn-4 deficiency on the hearing organ of the mouse. About one third of heterozygous female mice revealed late-onset profound deafness at the age of 1 year. Furthermore, in these deafened heterozygotes, characteristic abnormalities in Reissner's membrane attachment and type II fibrocytes in the suprastrial zone became evident under light microscope, similar to homozygous female mice. A significant reduction in the immunoreactivity of connexin 26 (Cx26), connexin 31 (Cx31), Na,K-ATPase and Na-K-Cl cotransporter in the spiral ligament fibrocytes was observed in aged heterozygotes showing late-onset profound deafness. The late-onset phenotype observed in heterozygous mutant mice, being consistent with the progressive deafness observed in human female heterozygotes, may be explained by alterations of the ion transport systems in the spiral ligament fibrocytes.