Background: Impairment of mitochondrial function plays an important role in obesity and the development of insulin resistance. The aim of this project was to investigate the mitochondrial DNA copy number in human omental adipose tissue with respect to obesity. Methods: The mitochondrial DNA (mtDNA) content per single adipocyte derived from abdominal omental adipose tissue was determined by quantitative RT-PCR in a group of 75 patients, consisting of obese and morbidly obese subjects, as well as non-obese controls. Additionally, basal metabolic rate and fat oxidation rate were recorded and expressed as total values or per kilogram fat mass. Results: MtDNA content is associated with obesity. Higher body mass index (BMI) resulted in a significantly elevated mtDNA count (ratio = 1.56; p = 0.0331) comparing non-obese (BMI < 30) to obese volunteers (BMI ≥ 30). The mtDNA count per cell was not correlated with age or gender. Diabetic patients showed a trend toward reduced mtDNA content. A seasonal change in mtDNA copy number could not be identified. In addition, a substudy investigating the basal metabolic rate and the fasting fat oxidation did not reveal any associations to the mtDNA count. Conclusions: The mtDNA content per cell of omental adipose tissue did not correlate with various clinical parameters but tended to be reduced in patients with diabetes, which may partly explain the impairment of mitochondrial function observed in insulin resistance. Furthermore, the mtDNA content was significantly increased in patients suffering from obesity (BMI above 30). This might reflect a compensatory response to the development of obesity, which is associated with impairment of mitochondrial functio