Copulatory behavior was studied in five groups of sexually experienced, gonadally intact male rats in which: (i) the nonsteroidal aromatase inhibitor Fadrozole (CIBA-Geigy CGS 16949A) was delivered bilaterally (0.756 microgram in 6.0 microliters saline in 24 h to each side) into the medial preoptic area (POM) together with normal saline given s.c. via osmostic minipumps (n = 10); (ii) normal saline was delivered bilaterally into POM together with the same dose of Fadrozole s.c. (n = 9); (iii) Fadrozole was delivered bilaterally into the lateral preoptic area together with normal saline given s.c. (n = 6); (iv) Fadrozole was delivered bilaterally into the cerebral cortex together with normal saline given s.c. (n = 6), and (v) unoperated controls (n = 14). Mounting and ejaculation were significantly decreased in rats receiving Fadrozole in POM compared with the behavior of rats in the other 4 groups. Few differences occurred between rats in the latter 4 groups, all of which continued to mate. 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 in gonadectomized controls and in some of the rats in groups i, ii and v. Since labeling of ERir neurons in rat brain with the H222 anti-ER antibody is reported to be inhibited by estrogen, it was used here to identify regions (with staining) where the aromatization of testosterone (T) into estradiol (E2) had been suppressed. Intense H222 ERir nuclear neuronal labeling was confined to the POM of males receiving Fadrozole in POM, and significantly more labeled neurons were found in the POM of these rats than in the POM of rats treated with saline in POM. In contrast, the ER-715 antibody, which is reported to stain neurons independently of hormonal status, labeled neuronal nuclei in hypothalamic and limbic regions of all groups, demonstrating the presence of ER. These findings show that conversion of T into E2 in the POM of the male rat is important for male rat copulatory behavior and that H222 ERir nuclear neuronal labeling can be used to identify the neurons in POM that were affected by Fadrozole.