Background: The purpose of our study was to investigate the association of C-peptide and steroid hormones in males and females with type 2 diabetes compared to controls. Methods: In 562 subjects, matched for age and body mass index (BMI), 126 female type 2 diabetic patients (known diabetes duration: 7.8+/-0.8 years, HbA(1c): 7.6+/-0.14%), 126 healthy female subjects, 155 male type 2 diabetic patients (known diabetes duration: 6.4+/-0.6 years, HbA(1c): 7.7+/-0.11%), and 155 healthy male controls, C-peptide levels and serum levels of steroid hormones (progesterone, cortisol, DHEAS, estradiol, and testosterone) were measured by immunometric assays. Ratios of steroid hormones were calculated to investigate shifts in steroidogenesis. Results: In female patients, testosterone was significantly higher than in controls (1.7+/-0.1 vs. 1.4+/-0.2 pmol/l; P<0.05), something that was also demonstrated for the ratio of testosterone/estradiol (P<0.05). In male patients, lower levels of testosterone (11.8+/-0.5 vs. 14.3+/-0.5 pmol/l; P<0.05) and higher cortisol levels (257.5+/-9.9 vs. 228.2+/-7.9 µmol/l, P<0.01) were found than in controls. The progesterone/DHEAS ratio (P<0.05) and the progesterone/testosterone ratio (P<0.001) were significantly higher and DHEAS/cortisol significantly lower in type 2 diabetic males than in controls. In a multiple linear regression analysis that controlled for age, BMI, C-peptide, HDL-cholesterol, and HbA(1c), testosterone was significantly and positively correlated with C-peptide levels in female (P<0.05) but not in male type 2 diabetic patients. Conclusions: Testosterone levels were higher in female patients than in controls and correlated with C-peptide levels while testosterone levels were lower in male patients than in controls and showed no correlation with C-peptide. A higher ratio of testosterone/estradiol in type 2 diabetic females and of progesterone/DHEAS and progesterone/testosterone in type 2 diabetic males than in controls may indicate gender-dependent shifts in steroidogenesis. Whether the shifts in steroidogenesis contribute to insulin resistance in diabetic patients should be the subject of further studies.