Majority of estrogen actions in the brain are mediated by estrogen receptor (ER) alpha which in turn is regulated by several factors like circulating levels of gonadal steroid hormones 17beta-estradiol and testosterone, sex and age of the organism. The expression of ERalpha is regulated through interaction between cis-elements of its promoter and proteins present in the nuclei. Here, we have used electrophoretic mobility shift assay (EMSA) to analyze the effect of age, sex, 17beta-estradiol, and testosterone on the binding of ERalpha promoter (-91 to +46 bp) to nuclear proteins from the mouse cerebral cortex. EMSA revealed the formation of three specific complexes in all groups. However, the intensity of these complexes varied as a function of age, sex and treatment with 17beta-estradiol and testosterone. Nuclear proteins from the cerebral cortex of both sexes showed reduced binding with promoter fragment in old mice. Further, competition analysis indicated stronger binding in females than males of both ages. The extent of binding was reduced by 17beta-estradiol and testosterone treatment in both ages and sexes. Thus, these findings demonstrate differential binding of nuclear proteins to mouse ERalpha promoter which may account for different functions of estrogen in the brain.