Brain aromatization of androgen to estrogen (E) and presence of estrogen-receptor (ER) containing cells (ERC) are required for the control of E-dependent neural events underlying male sexual behavior. We examined whether (a) numbers of ERC and steroid-inducible aromatase activity are codistributed and directly correlated in the same brain areas of individual sexually active male doves and whether, (b) distribution of ERC is altered by a change in E formation in preoptic areas known to be involved in control of male behavior. To allow spatial correlation between ERC and aromatase activity, a new approach was used that combined in vitro measurement of aromatase activity (stereospecific formation of <sup>3</sup>H<sub>2</sub>O from [1β-<sup>3</sup>H]-testosterone) in microdissected brain areas of one side of the brain and immunocytochemical localization of ERC (ER antibody H222Spy) in homologous contralateral areas of the same coronal brain section. The relationship between ERC and aromatase activity differs according to brain area in sexually active males: (a) large populations of ERC in preoptic areas, notably in nucleus preopticus medialis (6.5 ± 0.8 ERC/5,000 µm<sup>2</sup>) and nucleus preopticus medialis, pars medianis (9.5 ± 1 ERC/5,000 µm<sup>2</sup>) are codistributed with high steroid-dependent aromatase activity (>100 fmol/mg tissue); (b) areas containing the nucleus interstitialis and ventromedial hypothalamic nuclei also have relatively high aromatase activity, but lower ERC density than POA; (c) the anterior hypothalamic nuclei have few ERC, but steroid-regulated aromatase activity; (d) the infundibular area contains elevated ERC and little steroid-inducible aromatase activity; (e) area basalis and neostriatum contain few or no ERC and no inducible aromatase activity. Castration of sexually active doves reduces aromatase activity in preoptic and posterior hypothalamic areas (by > 75%) to basal levels, but does not affect the distribution or number of ERC in brain areas containing steroid-regulated aromatase activity, notably in the preoptic area. The results show that steroid regulation of aromatase occurs in brain loci with high numbers of ERC. We suggest that steroid-inducible aromatase activity and ERC are codistributed in areas controlling male sexual behavior; thus the formation and action of E may occur in the same area. Regulation of the aromatase activity and supply of E, but not of number of cells containing ER, is one mechanism which accounts for changes in action of testosterone on estrogen target sites in the male brain.