Testosterone is converted to estradiol in specific regions of the primate brain and accumulates as such in the nuclei of cells in hypothalamus, preoptic area, and amygdala. To locate more precisely those neurons in which nuclear estrogen receptors were occupied by estrogenic metabolites of testosterone, we injected 8 castrated male rhesus monkeys with [3H]-estradiol. Four were injected with oil for control purposes, and 4 were pretreated for 3 days with 2 mg/day testosterone propionate. This dose raised plasma testosterone levels into the high physiological range for intact males. After 60 min, brains were rapidly removed, the levels of [3H]-estradiol in nuclei were measured in the right halves of the brains by high-performance liquid chromatography, and labeled neurons were located in the left halves by autoradiography. Compared with the 4 control animals, nuclear levels of [3H]-estradiol in testosterone-treated males were reduced by 77% in the hypothalamus (p< 0.001), by 93% in the preoptic area (p< 0.001), and by 90% in the amygdala (p< 0.05). In autoradiograms from testosterone-treated males, the labeling of neurons was reduced by 72–96% in most of the regions in which the control males showed high percentages of labeled cells. However, there were only small reductions in the number of labeled neurons in lateral septum (by 31%) and arcuate nucleus (by 23%). These two regions, therefore, contained estrogen receptors that were not blocked by pretreatment with testosterone. The simplest explanation for these results is that estrogenic metabolites of testosterone prevented the uptake of [3H]-estradiol by prior occupation of estrogen receptor sites. The rather precise neuroanatomical localization of the effects pointed to the existence of two populations of estrogen target neurons in the primate brain depending on the presence or absence of local aromatase activit