Estrogen receptors (ERs) alpha and beta are very similar estrogen-binding proteins, perhaps gene duplication products, which act as ligand-dependent transcription factors. While the estrogenic regulation of ERalpha has been well documented, little is known about how estrogen regulates ERbeta and whether ERalpha plays a role in the expression and estrogenic regulation of ERbeta. In the present study, we examined the effects of gonadectomy and estrogen replacement on ERbeta immunoreactivity (ir) in wild-type (WT) and ERalpha knockout (alphaERKO) adult male mice in six brain regions, the medial preoptic area (MPOA), the bed nucleus of the stria terminalis (BNST), the paraventricular nucleus (PVN), the ventromedial nucleus of hypothalamus (VMH), the medial amygdala nucleus (MeAMY) and the dorsal raphe nucleus (DRN). Mice were divided into four different treatment groups: gonadally intact, gonadectomized (GDX), GDX+short-term treatment with estrogen (s.c. injection of estradiol benzoate (EB), 5 microg, at 48 h before perfusion) or GDX+long-term treatment with estrogen (implant of an EB pellet, 2.5 microg/day, for 10 days). In intact alphaERKO mice, the number of ERbeta expressing cells was significantly decreased in the MPOA and increased in the BNST, compared to WT mice. Both in the MPOA and BNST, steroid hormone regulation of ERbeta protein (an increase by GDX and a decline to intact levels by EB) was found only in WT, not in alphaERKO mice. In the VMH, GDX significantly increased the number of ERbeta ir expressing cells in both genotypes. EB treatment tended to decrease the number of ERbeta ir cells in WT mice, whereas EB treatment tended to increase ERbeta ir cell counts in alphaERKO mice. No effects of GDX or EB treatment were found in the DRN and MeAMY regardless of genotype. These results suggest that gonadal steroid hormones may regulate ERbeta protein in male mice and ERalpha may be involved in the expression and regulation of ERbeta in a region-specific manner.