To examine the frequency of the 961delT mitochondrial point mutation, considered to be associated with aminoglycoside-induced hearing loss, restriction fragment length polymorphism (RFLP) analysis was performed in (1) 334 unrelated sensorineural hearing loss (SNHL) patients and (2) 56 patients with aminoglycoside antibiotic injection history. Approximately 2% of the SNHL patients had the 961delT mutation, raising the possibility of a relatively high prevalence of this mutation among hearing impaired populations. However, the following findings cast doubt on whether this mutation is truly associated with hearing loss: (1) a similar frequency found in the control subjects, (2) hearing loss that was not segregated within the families, (3) rates of heteroplasmy and aging that were not correlated with the severity of hearing loss, and (4) a low prevalence among the aminoglycoside-induced hearing loss patients (1/56=1.8%). The present analysis did not agree with the concept that the 961delT mutation causes aminoglycoside-induced hearing loss.