Estrogen and progesterone interact in the regulation of various brain functions including mechanisms controlling gonadotropin secretion and female sexual behavior. Various methods have been used to map the regions where these hormones act and where specific receptors can be detected. However, it remains unknown if both steroids act on the same neuron or if there are neural populations responding to either one or the other of these hormones To answer this question, we used various immunocytochemical procedures to detect estrogen and progesterone receptors (ER-IR and PR-IR) on the same histological section, taking advantage of the fact that anti-ER monoclonal antibodies were raised from rats whereas anti-PR monoclonal antibodies were raised from mice. Initial experiments showed that the number of cells displaying ER and PR immunoreactivity changed with hormonal treatment. Prolonged treatment of ovariectomized guinea pigs with high doses of estradiol benzoate (EB) (10 or 15 micrograms/day for 4 or 6 days) increased the number of PR-positive cells in the preoptic area and the hypothalamus. Inversely, with this estrogen priming regimen the number of ER-positive cells decreased, as did the immunostaining intensity in their nuclei. With a lower dose of estrogen administered for a shorter period (2-microgram dose of EB for 2 days) the two receptor populations could be easily observed. Under these hormonal conditions, the sequential procedure revealed that the vast majority of cells containing ER-IR in the preoptic area (nucleus preopticus periventricularis, medialis) and the mediobasal hypothalamus (nucleus periventricularis, arcuatus, ventrolateral part of nucleus ventromedialis and premamillaris) showed PR immunoreactivity. This was true regardless of the order in which dual immunocytochemical staining was performed. There were, however, a few ER-positive/PR-negative cells in these regions. However, only ER-positive neurons were found in the n. interstitialis stria terminalis, the n. amygdaloideus medialis and the n. supraopticus. Thus, in guinea pigs receiving moderate doses of estrogen, all PR-positive cells contain ER-IR whereas there is a population of ER-positive cells which are devoid of PR-IR.