The H222 and ER-715 anti-estrogen receptor (ER) antibodies were used to examine the distribution of ER immunoreactive (ERir) neurons in hypothalamic and limbic sites of: (i) castrated male rats; (ii) castrated males implanted s.c. with silastic capsules containing testosterone (T), and (iii) castrated males receiving T together with 0.25 mg/kg/day of the nonsteroidal aromatase inhibitor, fadrozole (CIBA-Geigy CGS 16949A), delivered s.c. by means of implanted osmotic minipumps. Because labeling of ERir neurons in rat brain with H222 anti-ER antibody is reported to decrease when estrogen is present, it was used here to determine whether or not estrogen derived from the aromatization of T would affect ERir neuronal labeling. Castrated males showed H222 ERir-positive neurons in the lateral septum, medial preoptic area, several subdivisions of the hypothalamus, amygdala, and bed nucleus of stria terminalis. In contrast, in T-treated castrates, H222 ERir labeling was either eliminated or greatly reduced in all brain areas with the exception of the lateral septum. In castrated male rats given T together with fadrozole, H222 ERir labeling was restored in all brain areas where it had been reduced by T treatment. The ER-715 antibody effectively labeled neurons in all brain regions independently of the treatment condition, indicating that ER was present in the brains of animals in all treatment groups. These findings point to functional differences in ER dynamics in brain areas implicated in the control of sexual behavior by male rats. Neurons in the lateral septum, where aromatase activity is thought to be low, may derive estrogen directly from the systemic circulation whereas those in other brain regions may derive estrogen locally from T by aromatization.