OBJECTIVE To solve a possible relationship between mtDNA mutation of tRNALYS(8344) and diabetes, we have surveyed the tRNALYS mutation, glucose intolerance, and insulin secretory capacity in a Japanese family with diabetes and myoclonic epilepsy with ragged-red fiber disease. Several lines of evidence suggested possible linkage between mtDNA mutation and diabetes (1–4). RESEARCH DESIGN AND METHODS DNA was isolated from peripheral lymphocytes. The polymerase chain reaction analysis for the tRNALYS(8344) mutation of the mtDNA was conducted as described by Larsson (5). Insulin secretory capacity was assessed by 24-h urinary C-peptide immunoreactivity response (CPR) excretion and plasma CPR to glucagon administration. RESULTS We identified seven subjects with the tRNALYS mutation as well as seven non-mutated members in the pedigrees. Oral glucose tolerance tests in the pedigree indicated that five of the mutated subjects were diabetic, one had impaired glucose tolerance, and one had normal glucose tolerance (NGT), whereas all nonmutated family members had NGT. The pedigree shows maternal transmission of diabetes and the tRNALYS mutation over three generations. Twenty-four-hour urinary excretion of CPR was significantly reduced in the mutant subjects (mean ± SD, 67.8 ± 79.2 nmol/day, n = 6, P < 0.001) compared with the nonmutant members (276.6 ± 41.8 nmol/day, n = 5) and the age-matched normal control subjects (263 ± 64.3 nmol/day, n = 12). Plasma CPR 6 min after glucagon injection demonstrated a marked reduction in the mutant subjects (3.68 ± 3.45 nmol/l, n = 5, P < 0.001) compared with the nonmutant members (19.4 ± 1.17 nmol/l, n = 5) and the normal control subjects (15.8 ± 3.81 nmol/l, n = 12). Bilateral neurosensory deafness was demonstrated in five of seven (71.4%) mutant subjects (five of five [100%] mutated diabetic patients), but not detected in six nonmutant members. CONCLUSIONS This observation is the first report of association of diabetes with the mitochondrial tRNALYS mutation. Insulin secretory capacity was significantly lower in the mutant members than in the nonmutated members. These findings suggest that the pancreatic β-cell secretory defect of insulin might be one of the phenotypes of the mitochondrial tRNALYS mutation.