Estrogen induction of cytosolic progestin receptors (CPRs) in the hypothalamus-preoptic area of the female guinea pig is correlated with facilitation of female sexual behavior and gonadotropin secretion by progesterone. The present study validated a CPR microassay and determined where, within microdissected areas of the hypothalamus-preoptic area of the female guinea pig, induction of CPRs by estradiol occurs. Ovariectomized adult guinea pigs were given 20 µg estradiol benzoate (EB) or oil vehicle for 3 successive days. CPRs were measured using the synthetic progestin [3H]-R5020. The highest basal (no estrogen treatment) level of CPRs was seen in the arcuate-median eminence (34.1 ± 3.7 fmol/mg). With EB treatment, the highest level of CPRs was again in the arcuate-median eminence (178.0± 12.0 fmol/mg). EB-treated females also had high CPR levels in the periventricular area (88.5 ± 10.8 fmol/mg) and the medial preoptic area (86.3 ± 9.3 fmol/mg). Moderate levels were seen in the ventromedial nucleus of the hypothalamus (32.7 ± 3.0 fmol/mg) and in the anterior hypothalamic nucleus (13.0 ± 2.1 fmol/mg), but these were not significantly different from the low levels in the medial amygdala (4.5 ± 1.2 fmol/mg) and in the dorsomedial nucleus of the hypothalamus (5.4 ± 1.1 fmol/mg) of EB-treated females. However, EB caused a significant induction over baseline levels not only in the arcuate-median eminence, periventricular area, and medial preoptic area, but also in the ventromedial nucleus of the hypothalamus and the anterior hypothalamic nucleus. EB did not increase CPRs in the medial amygdala or the dorsomedial nucleus of the hypo