The orphan nuclear estrogen receptor-related receptor (ERR) alpha is expressed by osteoblastic cells, is known to transactivate at least one osteoblast-associated gene osteopontin (OPN) and plays a functional role in osteoprogenitor cell proliferation and differentiation. To dissect further the role of ERR-alpha in bone formation, we compared its expression to that of the estrogen receptor (ER) alpha and ER-beta in rat calvaria (RC) and fetal tibia in vivo and in RC and rat bone marrow (RBM) cells in vitro. We found that ERR-alpha is highly and widely expressed in most, if not all, cells in RC cell cultures from early proliferation stages through mineralized nodule formation; ER-alpha was localized similarly but at lower levels and ER-beta, although present, was barely detectable. These patterns of expression in vitro correlated with what we observed in vivo in sections of 21-day fetal RC, in which ERR-alpha appeared to be more highly expressed than either of the ERs. Interestingly, ERR-a also is highly expressed in RBM cells, while ER-alpha and ER-beta mRNA is expressed, but at lower levels. Moreover, we found that ERR-alpha, ER-alpha, and ER-beta were all expressed in osteoblasts in fetal and adult tibia whereas they were expressed differentially in calvaria in vivo in subsets of osteoblasts, supporting the hypothesis that ERR-alpha may interact with one or both of the ERs in those osteoblasts in which they are coexpressed and that all three receptors may be required for bone formation but at different times and for different functions.