Background: The aim of this study was to investigate the effect of hypothyroidism on lipid peroxidation and the antioxidant profile, as well as to evaluate the interaction between thyroid hormones and biomarkers of oxidative stress in patients with overt hypothyroidism. We also evaluated the influence of cholesterol concentrations on biomarkers of oxidative stress in these same patients. Methods: Total cholesterol (TC), high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, triglycerides, thiobarbituric acid reactive substances (TBARS), superoxide dismutase (SOD), catalase (CAT), and vitamin E were measured in 20 subjects with overt hypothyroidism (OH) and 20 controls. Results: TC, LDL cholesterol, triglycerides, TBARS, SOD, CAT, and vitamin E were significantly higher in the OH group. Significant correlation was observed for TSH and SOD, CAT, vitamin E and TBARS. Correlation was observed for triiodothyronine (T3) and SOD, CAT, vitamin E and TBARS. Significant correlation was also observed for free thyroxine and vitamin E and TBARS. However, correlation between T3 and CAT remained significant after controlling for TC concentrations. Conclusions: Overt hypothyroidism is associated with an increase in oxidative stress, and hypercholesterolemia has a stronger influence on development of oxidative stress in hypothyroid conditions compared with thyroid hormones. Clin Chem Lab Med 2010;48:1635–9.