Previous studies have shown that [3H]estradiol is the major nuclear metabolite of [3H]testosterone in the hypothalamus, preoptic area and amygdala of the male rhesus monkey, and it has been proposed that some of the stimulatory effects of testosterone on male sexual behavior may be mediated by estradiol-concentrating neurons. To map these neurons and identify metabolites, 4 castrated male rhesus monkeys were each injected with 0.47 mCi/kg [3H]estradiol, and the brains were removed 60 min later. Left halves were frozen for thaw-mount autoradiography, and right halves were used to isolate cell nuclei from different brain regions. A fifth animal was used to validate the methodology. Radioactive steroids were identified and measured by high performance liquid chromatography. Brain areas previously shown to contain labeled neurons after [3H]testosterone administration were also labeled after [3H]estradiol, including medial preoptic nucleus (n.), anterior hypothalamic area, bed n. of stria terminalis, ventromedial n., premammillary n., and corticomedial amygdala. Some areas not labeled after [3H]testosterone were labeled after [3H]estradiol, including lateral septal n., arcuate n., paraventricular n., claustrum, entorhinal cortex, and spinal cord. The pars distalis of the pituitary gland was heavily labeled. Most (83%) of the radioactivity in cell nuclei was [3H]estradiol while [3H]estrone was a major metabolite (25%) in supernatants. In the brain, the highest nuclear concentration of [3H]estradiol was in the hypothalamus (249.2 ± 20.0 fmol/mg DNA), although in the previous experiments with [3H]testosterone, the highest nuclear concentrations of [3H]estradiol were found in the amygdala. We interpret these results to indicate that local metabolic differences in the brain may underlie some of the different behavioral effects of gonadal steroids in primates