Estrogen receptor-related orphan receptor α1 is a member of the steroid/thyroid nuclear receptor superfamily. We have previously cloned the human estrogen receptor-related orphan receptor α1 (hERR α1) cDNA and demonstrated that it enhances estrogen responsiveness of the lactoferrin gene promoter in transfected human endometrial carcinoma cells. In the present study, we used the hERR α1 cDNA as a probe and isolated the mouse homologue of ERR α1 from the cDNA libraries of the brain and kidney. Sequence comparison between human and mouse ERR α1 (mERR α1) revealed that the homologies are 89% in nucleotides and 97% in amino acids. By electrophoresis mobility shift assay, we showed that the glutathione S-transferase-mERR α1 fusion protein produced in a bacterial system bound to the human ERR α1 DNA-binding element. Mouse uterine nuclear extract also interacted with this DNA element and produced three complexes in the mobility shift assay, one of which was supershifted by the hERR α1 antiserum. A 2· 2 kbp transcript was detected by Northern analysis in all adult mouse tissues tested; however, large variations in the amount of ERR α1mRNA were found among them. Multiple immunoreactive forms of mouse ERR α1 were detected by Western analysis in non-reproductive tissues, whereas a major 53 kDa protein was found in reproductive tissues such as uterus, cervix and vagina. Diethylstilbestrol (DES) stimulated the expression of ERR α1mRNA in the uterus of 19-day-old mouse. We showed that DES and estradiol, but not progesterone or dexamethasone, enhanced the level of immunoreactive ERR α1 in the mouse uterus. These results demonstrated that the ERR α1 is an estrogen-responsive gene in the mouse uterus and provides a model system with which to study the biological roles of this nuclear orphan receptor.