We studied the expression of estrogen-related receptor ERR-1 during mouse embryonic development. ERR-1 mRNA is present in bones formed by both the endochondral and intramembranous routes, and the onset of its expression coincides with bone formation. By RT-PCR experiments, we found that ERR-1, but not the related receptor ERR-2, is expressed in osteoblastic osteosarcoma cell lines as well as in primary osteoblastic cell populations derived from normal human bone. By gel shift analysis we found that ERR-1 binds as a monomer specifically to the SFRE sequence (SF-1-responsive-element; TCAAGGTCA). Mutation analysis revealed that both the core AGGTCA motif and the TCA 5'-extension are required for efficient ERR-1 binding. In transient transfection assays, ERR-1 acts as a potent transactivator through the SFRE sequence. This effect is cell-specific since ERR-1 activates transcription in the rat osteosarcoma cell line ROS 17.2/8 as well as in HeLa, NB-E, and FREJ4 cells but not in COS1 and HepG2 cells. Notably, the osteopontin (a protein expressed by osteoblasts and released in the bone matrix) gene promoter is a target for ERR-1 transcriptional regulation. Our findings suggest a role for ERR-1 in bone development and metabolism.