Estrogen receptor β (ERβ) is a novel steroid receptor that is expressed in rat prostate and ovary. We have cloned the mouse homolog of ERβ and mapped the gene, designated Estrb, to the central region of chromosome 12. The cDNA encodes a protein of 485 amino acids that shares, respectively, 97% and 60% identity with the DNA- and ligand-binding domains of mouse (m) ERα. Mouse ERβ binds to an inverted repeat spaced by three nucleotides in a gel mobility shift assay and transactivates promoters containing synthetic or natural estrogen response elements in an estradiol (E₂)-dependent manner. Scatchard analysis indicates that mERβ has slightly lower affinity for E₂ [dissociation constant (K_d) = 0.5 nm] when compared with mERα (K_d = 0.2 nm). Antiestrogens, including 4-hydroxytamoxifen (OHT), ICI 182,780, and a novel compound, EM-800, inhibit E₂-dependent transactivation efficiently. However, while OHT displays partial agonistic activity with ERα on a basal promoter linked to estrogen response elements in Cos-1 cells, this effect is not observed with mERβ. Cotransfection of mERβ and H-Ras^V12 causes enhanced activation in the presence of E₂. Mutagenesis of a serine residue (position 60), located within a mitogen-activated protein kinase consensus phosphorylation site abolishes the stimulatory effect of Ras, suggesting that the activity of mERβ is also regulated by the mitogen-activated protein kinase pathway. Surprisingly, the coactivator SRC-1 up-regulates mERβ transactivation both in the absence and presence of E₂, and in vitro interaction between SRC-1 and the ERβ ligand-binding domain is enhanced by E₂. Moreover, the ligand-independent stimulatory effect of SRC-1 on ERβ transcriptional activity is abolished by ICI 182,780, but not by OHT. Our results demonstrate that while ERβ shares many of the functional characteristics of ERα, the molecular mechanisms regulating the transcriptional activity of mERβ may be distinct from those of ERα.