We have earlier described hyperprolactinemia in some patients presenting inner ear dysfunction. However, in that study it was not possible to determine whether hyperprolactinemia was a cause or an effect of the symptoms. In order to investigate the effect of hyperprolactinemia on inner ear function we first developed the model of hyperprolactinemia in estrogen-primed Fischer 344 rats and then performed functional studies on pigmented guinea pigs. Hyperprolactinemia induced, after two months, a hearing loss of around 30-40 dB across all frequencies as indicated by the compound action potential audiogram. During the third month the hearing loss continued to deteriorate. The threshold shifts were more substantial in males than females. Observations under the dissection microscope revealed bone dysmorphology of the bulla as well as the cochlea. Light microscopy observations of cryostat sections confirmed bone-related pathology of the bony cochlear bulla and the cochlear wall and revealed morphopathology of the stria vascularis and spiral ligament. Scanning electron microscopy revealed loss of hair cells and stereocilia damage, in particular, in the upper three cochlear turns and the two outermost hair cell rows. The data provide the first evidence of otic capsule and hair cell pathology associated with estrogen-induced prolonged hyperprolactinemia and suggests that conditions such as pregnancy, anti-psychotic drug treatment, aging and/or stress might lead to similar ear dysfunctions.