In this study we characterized clinically and evaluated molecularly a large family with maternally inherited hearing impairment. Relatives were evaluated audiologically and clinically, the most likely pattern of inheritance was deduced, and molecular DNA analysis for the known mitochondrial mutations associated with hearing impairment was performed. Clinical examination of several relatives showed a normal general state of health, but in 14 of the members tested variable degrees of sensorineural hearing loss were noted. The pedigree was established and demonstrated a clear pattern of maternal inheritance, with 34 of 38 offspring of deaf mothers being hearing impaired, but none of 22 offspring of deaf fathers having any hearing impairment. Since by far the most likely explanation of such a maternal inheritance pattern is a mitochondrial mutation, molecular testing for the three known mitochondrial mutations, A1555G, A7445G, and Cins7472, was performed on 27 of the relatives. All of the individuals tested had the normal sequence at the sites tested. This family with nonsyndromic sensorineural hearing loss has an inheritance pattern strongly suggestive of a mitochondrial mutation. However, molecular testing for the three known mitochondrial mutations associated with nonsyndromic hearing impairment was negative, implying that additional molecular defects can lead to the same phenotype. The search for this novel molecular defect is underway.