There are well known differences between males and females in hearing. In the present study, the role of estrogen receptor-b (ER-b; listed as ESR2 in the MGI Database) in hearing was investigated by comparing hearing and morphology of the inner ear in ER-b knock-out mice (ER-b) with that of wild-type (WT) littermates. Hearing was analyzed with auditory brainstem response audiometry at 3 and 12 months. The ER-b K/K mice were deaf at 1 year of age, and the morphological analysis showed absence of hair cells and loss of the whole organ of Corti initiated in the basal turn of the cochlea. Furthermore, in ER-b, but not in WT mice, the spiral ganglion was lacking many of its neurons.Immunostaining showed the presence of both ER-a (listed as ESR1 in the MGI Database) and ER-b in the nuclei of some neurons in the inner ear in WT mice, but no ER-b was found in the ER-b K/K mice as expected. ER-a staining was predominant in the nuclei of large neurons and ER-b in nuclei of small neurons and fibroblasts. These results reveal that both ERs are present in the inner ear at specific localizations suggesting subtype-specific functions. It is concluded that ER-b is important for the prevention of agerelated hearing loss. These findings strengthen the hypothesis that estrogen has a direct effect on hearing functions.