We report on a new maternally-inherited syndrome characterized by a combination of sensorineural hearing loss, ataxia and myoclonus in a large kindred from Sicily. Hearing loss was the most widespread and sometimes the only symptom found in family members. Sequence analysis of the mitochondrial DNA regions encompassing the tRNA genes revealed the presence of a heteroplasmic insertion at nucleotide position 7472. The insertion adds a seventh cytosine to a six-cytosine run that is part of the mitochondrial tRNASer(UCN) gene. Conformational analysis showed that this mutation is likely to alter the structure of the T psi C loop in the tRNASer(UCN) clover leaf secondary structure. Moreover, the degree of heteroplasmy in blood and muscle was correlated with the clinical phenotype, and homoplasmic mutant hybrids showed decreased complex I activity, low oxygen consumption and high lactic acid output, indicating faulty oxidative phosphorylation. Finally, mutation was absent in 381 unrelated maternal lineages, suggesting specific segregation with the disease. We propose that the C7472 insertion-mutation is pathogenic, and etiologically related to hearing loss and other symptoms that define a novel maternally-inherited clinical entity.