Intracranial implants of estradiol benzoate (EB) were used to explore sites of action for the activation of masculine copulatory behavior in castrated male rats. In experiment 1, 27 or 30 gauge implants were placed unilaterally throughout the hypothalamus and preoptic area. Dihydrotestosterone (DHT) was administered subcutaneously (s.c.) to each male. 25 of 42 males with EB implants located in the medial anterior hypothalamic-preoptic area (AHPOA) exhibited ejaculatory patterns on at least 40% of the post-implantation tests. In contrast, only 4 of 18 of the males with implants located posterior to the anterior hypothalamic area displayed this level of response. This difference in the proportion of animals responding for each implant group was statistically significant. Seminal vesicle weights were not correlated with the occurrence of copulation. In experiment 2, either EB-filled or empty cannulae were placed unilaterally in the AHPOA. Half of the males of each group also received DHT s.c. Intracranial EB in conjunction with DHT s.c. resulted in a significantly greater frequency of ejaculatory response than any other treatment. Despite significant differences among the groups in seminal vesicle weights, there was no correlation with occurrence of ejaculatory behavior. These results are consistent with the hypothesis that testosterone (T) exerts its effects upon masculine sexual behavior via conversion to estradiol (E2); and that the principal neural site of action is the AHPOA.