Oxysterols have cytotoxic effects and contribute to the development of atherosclerosis. To examine association between 7-ketocholesterol and diabetes mellitus, and other coronary risk factors, we developed a reliable quantitative method to measure serum 7-ketocholesterol (s-7KCHO) and studied s-7KCHO in patients with type 2 diabetes mellitus (T2DM). The s-7KCHO was detected by gas chromatography-mass spectrometry assay. The s-7KCHO was significantly higher in patients with T2DM (n=137, 33.8 ng/ml) compared to non-diabetic healthy subjects (n=89, 16.1 ng/ml). Patients with T2DM were divided into two groups with two or more than two risk factors (defined as multiple risk factors group) and with zero or one risk factor (non-multiple risk factors group). The s-7KCHO was significantly higher in multiple risk factors group (39.5 ng/ml) compared to non-multiple risk factors (30.1 ng/ml). Among patients with multiple risk factors group, s-7KCHO was significantly higher in patients with high low-density lipoprotein cholesterol (LDL-C) levels (45.1+/-5.9 ng/ml) compared to those with normal LDL-C levels (35.3+/-7.0 ng/ml). Furthermore, s-7KCHO increased according to the number of concurrent coronary risk factors. These results suggest that serum 7-ketocholesterol levels may depend on the multiple risk factors and serum LDL-C levels.