The amount of cytoplasmic receptor for the steroid hormone, estradiol (E2), was determined in 46 nuclei and subdivisions of rat brain. Individual nuclei were removed from 300-micrometers frozen sections according to the punch-out method of Palkovits (Palkovits, M. (1973) Brain Res. 59: 449-450), and the content of E2 receptor was measured with a sensitive radioligand binding method. Cytoplasmic receptors for E2 were distributed heterogeneously throughout the rat brain. The highest level of receptor (40 fmol/mg of protein) was found in the periventricular nucleus of the preoptic area, while low (1 fmol/mg) but detectable levels of receptors were found in such limbic regions as the nucleus of the diagonal band, the olfactory tubercle, and the cingulate cortex. Regions that were devoid of detectable receptor included the medial septum, the parietal cortex, and the ventral thalamus. Our results support the notion that E2 influences reproductive behavior and neuroendocrine function by binding to receptors in discrete areas of the brain and provide the first quantitative map of E2 receptors in individual rat brain nuclei.