Arterial calcification has prognostic significance for cardiovascular outcomes, but its pathogenesis remains unclear. Calcification increases with age, but its prevalence in men suggests hormonal influence. In this study we analyzed the effect of exogenous androgens on calcification of advanced atherosclerotic lesions in the arterial tree of gonadally intact 34-wk-old male and female apolipoprotein E-null mice. Testosterone (T) increased calcification 3-to 4-fold (P < 0.05) in lesions of the innominate artery and aortic sinus. A nonaromatizable androgen, dihydrotestosterone, also increased lesion calcification in the innominate artery (2.4-fold, P < 0.05) but not the aortic sinus. The androgen-induced effects were independent of sex and occurred despite corresponding reductions in plaque area, the latter correlating inversely with increased serum high-density lipoprotein cholesterol levels. Androgen-induced calcification in the innominate artery was observed with up-regulation of local androgen receptor (AR) expression in response to T and dihydrotestosterone for both males and females but neither androgen influenced innominate artery estrogen receptor (ER)-α or β expression in either sex. Conversely, T-induced calcification in the aortic sinus was associated with down-regulation of ERα but not ERβ expression in both sexes, whereas androgen- induced AR expression was increased in female but decreased in male mice. This study demonstrates for the first time that calcification of advanced atherosclerotic lesions is an androgen - sensitive process and postulates potential roles for both AR- and ER-mediated pathways in androgen-induced vascular calcification. We demonstrate a novel direct link between vascular calcification and the major male hormone, T, uncoupled from conventional relationships with plaque growth and lipid levels.