In female rats, the onset of reproductive behavior depends on the sequential presence of estradiol followed by progesterone. Although treatment with high doses of estradiol has been shown to increase immunostaining for the Fos protein, an immediate early gene product that is expressed upon cellular activation, another report conflicts with this finding. However, the previous reports agree that subsequent treatment with progesterone has no apparent effect on Fos expression. In order to resolve this discrepancy and investigate possible effects of progesterone, we used Fos immunocytochemistry combined with computer- aided image analysis. In experiment one, we found that treatment with 5 micrograms of estradiol increased Fos immunoreactivity (Fos-IR) within a section of the medial preoptic area and the dorsal medial hypothalamus. Subsequent treatment with 500 micrograms of progesterone 1 hr before perfusion increased the intensity of the immunostaining within the medial preoptic area and the dorsal medial hypothalamus, although it had no significant effect on Fos-IR cell number. In experiment 2, a lower concentration of Fos antiserum was used in order to diminish the immunostaining sensitivity to a level in which no increase of Fos-IR cell number was observed after treatment with estradiol. Under these immunocytochemical conditions, subsequent treatment with progesterone increased the number of Fos-IR cells in the medial preoptic area, the dorsal medial hypothalamus and the steroid receptor-rich area lateral to the ventromedial hypothalamus. Thus, treatment with behaviorally effective doses of both estradiol and progesterone induces Fos expression in localized regions of female rat brain.