Regions of the brain that concentrate estrogen and progesterone are thought to regulate female sexual behavior by altering gene expression and neural sensitivity to afferent stimulation. We used immunocytochemistry and in situ hybridization to examine c-fos gene expression within estrogen-concentrating regions of the forebrain following various types of sexual stimulation with or without hormone treatment. Ovariectomized rats received injections of estradiol benzoate 48 h and progesterone 4 h before testing. Control rats that had been ovariectomized at least 5 months before testing did not receive hormone treatment. Rats were then either placed into bilevel testing chambers with sexually vigorous males, received manual stimulation of the flanks, received vaginocervical stimulation with a glass rod, or were left in their home cages. Copulation with intromission and ejaculation in hormone-treated rats, or stimulation of the vaginal cervix in both hormone-treated and control rats, produced a dramatic induction of c-fos mRNA and Fos-like immunoreactivity in estrogen-concentrating regions, such as the lateral septum, medial preoptic area, bed nucleus of the stria terminalis, paraventricular nucleus of the hypothalamus, ventromedial hypothalamus, lateral habenula, and medial amygdala, in addition to regions that do not readily concentrate estrogen, such as the neocortex, thalamus, and striatum. Mechanical stimulation of the flanks produced a smaller induction of Fos in these rats, whereas hormone treatment alone had no effect. These data demonstrate that afferent sensory stimulation, but not estrogen or progesterone, regulates c-fos gene expression within different estrogen-concentrating and non-concentrating regions of the female rat forebrain.