A fluorescent immunocytochemical technique was developed to determine if cells in the guinea pig hypothalamus and preoptic area that contain estradiol-induced progestin receptors also contain estrogen receptors. With this technique little or no progestin receptor-immunoreactivity (PR-IR) was observed in the absence of estrogen treatment in ovariectomized guinea pigs. As has been reported previously, priming with estradiol caused a large increase in the concentration of PR-IR cells in discrete regions of the hypothalamus and preoptic area, primarily in the arcuate nucleus, ventrolateral area of the hypothalamus, periventricular preoptic area, medial preoptic nucleus, medial preoptic area, anterior hypothalamic nucleus and anterior hypothalamus. A range of lightly to intensely labeled estrogen receptor-immunoreactive (ER-IR) cells were observed in high concentration in each of these areas, as well as in some areas in which no PR-IR cells have been identified, such as the amygdala. PR-IR was only observed in cells that also had ER-IR. In some areas such as the ventrolateral hypothalamic area and arcuate nucleus, nearly all medium to highly-fluorescent ER-IR cells also contained estradiol-induced PR-IR, while in the amygdala no PR-IR was observed despite a high concentration of ER-IR cells. These results confirm the hypothesis that progestin receptors are produced in estrogen receptor-containing cells in the brain, and they suggest that these cells are the sites where estradiol and progesterone act to influence behavior and physiology.