Interactions between glutamate and gonadal steroids are involved in the regulation of limbic and hypothalamic functions. We hypothesized that hormonal signals affect excitatory neurotransmission by regulating the expression of glutamate receptors (GluR) in limbic and hypothalamic regions. To test this hypothesis, first, the coexpression of dl-alpha-amino-3-hydroxy-5-methyl-4-isoxazone-propionate (AMPA) GluR1, GluR2/3, and androgen receptors or estrogen receptors was revealed in the same cells of septal, amygdaloid, and hypothalamic areas by double immunocytochemistry. The highest incidence of co-localization was detected in hypothalamic regions. To demonstrate a regulatory role of testosterone or estradiol on AMPA receptor expression, the hormonal milieu of male and female rats was manipulated by gonadectomy and hormonal treatment. GluR1 and GluR2/3 expression was assessed by Western blots. Statistical analysis demonstrated that testosterone and estradiol have a stimulatory influence on the expression of AMPA receptors in the hypothalamus. The regulatory effect of estradiol on AMPA receptors was found to be site and gender specific: after estradiol treatment, samples taken from the hypothalamus contained increased levels of GluR1 and GluR2/3, whereas in the septum, bed nucleus and amygdala, no changes could be detected. Furthermore, the increase in hypothalamic GluR 2/3 levels was two times higher in females, compared with males, whereas the changes in hypothalamic GluR 1 levels showed no sex differences. Our results support the hypothesis that the interaction between gonadal steroids and glutamate involves hormone regulation of GluR. This mechanism seems to be gender and site specific, suggesting that excitatory neurotransmission and related physiological mechanisms also may be distinctly different in males and females.