Klotho-deficient mice exhibit a premature aging syndrome, a feature of which is mild hearing loss. In the present study, the hearing phenotype of Klotho mice was characterized to better determine how well this phenotype resembles presbycusis in humans. It was demonstrated that Klotho animals have auditory-evoked brainstem response (ABR) threshold shifts of 14-18 dB in response to pure tone stimuli of 4, 8, 16 and 32 kHz, and similarly, in response to clicks; however, cochlear histology and spiral ganglion neuron density appeared normal in these mice. It was further demonstrated that a vitamin D-deficient diet normalizes serum calcitriol (1,25(OH)2D3) levels and prevents hearing loss in Klotho mice. It is concluded that hearing loss in Klotho mice is caused by elevated renal 1a-hydroxylase expression and consequent excessive production of calcitriol. These findings implicate the vitamin D metabolic pathway in hearing loss and pose questions as to the mechanism by which elevated calcitriol levels mediate such hearing loss. ? 2010 Elsevier B.V.