Both the ahl allele of Cdh23 and the null mutation of Sod1 have been shown to contribute to age-related hearing loss (AHL) in mice, but mixed strain backgrounds have confounded analyses of their individual and combined effects. To test for the effects of Sod1 deficiency independently from those of Cdh23(ahl), we produced mice with four digenic genotypes: Sod1(+/+)Cdh23(ahl)(/ahl), Sod1(+/+)Cdh23(+/+), Sod1(-/-)Cdh23(ahl)(/ahl), and Sod1(-/-)Cdh23(+/+), all on a uniform C57BL(/)6J strain background. We assessed hearing loss by ABR threshold measurements and evaluated cochlear pathologies in age-matched mice of each digenic combination. ABR analysis showed that Sod1(+/+)Cdh23(+/+) mice retain normal hearing up to 15 months of age and that hearing loss of Sod1(+/+)Cdh23(ahl)(/ahl) mice is more age and frequency dependent than that of Sod1(-/-)Cdh23(+/+) mice. ABR results also showed that mice with both gene mutations (Sod1(-/-)Cdh23(ahl)(/ahl)) exhibit the earliest onset and most severe hearing loss, greater than predicted for strictly additive effects. Histological analysis of cochleas showed that hair cell lesions are most severe in Sod1(-)(/-)Cdh23(ahl)(/ahl) mice followed closely by Sod1(+)(/+)Cdh23(ahl)(/ahl) mice and much smaller in Sod1(-)(/-)Cdh23(+)(/+) and Sod1(+)(/+)Cdh23(+)(/+) mice. Despite extensive damage to cochlear hair cells, vestibular hair cells appeared remarkably normal in all strains. Although both Sod1(-/-) and Cdh23(ahl)(/ahl) genotypes had strong effects on hearing loss, the Cdh23(ahl/ahl) genotype was primarily responsible for the increase in hair cell loss, suggesting that the two mutations have different underlying mechanisms of pathology.