Objective: To investigate whether circulating levels of testosterone (total, bioavailable), estradiol (total, bioavailable), and DHEA sulfate (DHEAS) are associated with fasting plasma homocysteine (tHcy) levels in middle-aged and elderly men. Design: A population-based sample of 400 independently living men between 40 and 80 years of age in a cross-sectional study. Methods: Total testosterone, sex hormone binding globulin (SHBG), and total estradiol were measured by RIA methods and bioavailable testosterone and estradiol were calculated. DHEAS was measured using an immunometric technique. Fasting homocysteine was measured by fluorescence polarization immunoassay. Anthropometric characteristics were also measured and two standardized questionnaires completed, including life-style factors and diet. Linear regression analysis adjusted for age, body mass index (BMI), creatinine clearance, and mean visceral fat was used to assess the association of endogenous sex hormones and fasting plasma homocysteine levels. Results: After adjustment for age, BMI, creatinine clearance, and mean visceral fat no statistically significant association was observed between testosterone (total, bioavailable), DHEAS, and estradiol (total, bioavailable)levels with natural log tHcy (β = - 2 × 10-3; 95% confidence intervals (CI) - 9 × 10-3; 5 × 10-3), (β= -4 × 10-3; 95%CI -18 × 10-3; 9 × 10-3), (β= 3 × 10-3; 95%CI - 6 × 10-3; 12 × 10-3), (β= -9.3 × 10-5; 95% CI - 1 × 10-3; 1 × 10-3), and (β=0.00; 95% CI - 3 × 10-3; 2 × 10-3) respectively. Additional adjustment for smoking, alcohol intake, daily physical activity, diabetes mellitus, and hypertension did not change these findings. Conclusion: The results of our study do not support a direct role for circulating sex hormone levels in the regulation of fasting plasma tHcy concentrations in middle-aged and elderly men.