Qualitative changes in mitochondrial DNA (mtDNA), such as mutations and deletions, have been implicated in the pathogenesis of diabetes mellitus. In addition to the qualitative changes, mtDNA is subject to quantitative changes, and is vulnerable to oxidative stress, resulting in both qualitative and quantitative changes. This study was performed to investigate whether quantitative changes in mtDNA occur in non-insulin-dependent diabetes mellitus (NIDDM) patients and also in pre-diabetic subjects. MtDNA content from peripheral blood was measured by slot-blot analysis in 55 NIDDM patients and 29 age- and sex-matched control subjects. We have also analysed the mtDNA copies by quantitative polymerase chain reaction (PCR) method in 23 pre-diabetic subjects who converted to diabetic in 2 years and 22 age- and sex-matched control subjects who remained non-diabetic. Mean mtDNA quantity measured by slot blot method was 35% lower in patients with NIDDM than in control subjects (12.3+/-8.1 vs. 19.1+/-8.2 AU/microg DNA; P < 0.05). MtDNA quantities did not correlate with age, body mass index, duration of diabetes or HbA1c levels. We have also found that the mtDNA copies in subjects who converted to diabetes in 2 years were lower than in controls even before the development of diabetes (102.8+/-41.5 vs. 137.8+/-67.7 copies/pg template DNA P < 0.05). Inverse correlations were noted between mtDNA content and baseline waist hip circumference ratio (WHR) (r = -0.31, P < 0.05), and fasting glucose level (r = - 0.35, P < 0.05), diastolic blood pressure (r = -0.36, P < 0.05), and WHR (r = -0.40, P < 0.01) after development of diabetes. In conclusion, we demonstrate that the content of mtDNA decreases in peripheral blood of patients with NIDDM and the lower mtDNA levels precede the development of diabetes.