The purpose of these experiments was to develop a method of isolation, amplification, and identification of cochlear mitochondrial DNA (mtDNA) from minute quantities of tissue. Additionally, studies were designed to detect mtDNA deletions (mtDNA del) from the cochlea that previously have been amplified from other organ systems and tissues. MtDNA del have been associated with many pathologies, including neurological disorders, sensorineural hearing loss, ischemia, cardiomyopathies, and aging. DNA was extracted from rat and human tissues, and polymerase chain reaction was used to amplify mtDNA sequences. A 360 base pair (bp) cytochrome-b gene product and the highly conserved ND1-16S ribosomal ribonucleic acid regions found only in mtDNA were amplified from all tissues. Preliminary studies have identified a 4834 bp mtDNA del in aged rats and a corresponding 4977 bp mtDNA del in aged humans. Additionally, preliminary results in human archival temporal bone studies reveal the presence of the 4977-bp mtDNA deletion in two out of three patients with presbycusis. The deletion was not evident in age-matched control patients without a history of presbycusis. This technique of mtDNA identification makes it possible to investigate specific mtDNA defects from a single cochlea, promoting the study of hereditary hearing loss and presbycusis at a molecular biologic level.