High-dose estrogen administration is known to induce new bone formation in mouse long bones. To study the role of regulatory proteins in this response, we examined associated changes in femoral messenger RNA (mRNA) for candidate factors. 17beta-estradiol (E2) 0.5 mg was administered to intact female mice by weekly injection, and Northern blot analysis was performed 1, 2, 4, 8, 12, and 16 days after the first injection. In contrast to other factors, an increase was observed in mRNA for bone morphogenetic protein-6 (BMP-6), which reached significance at day 8 and subsequent time-points. Estrogen-induced changes in BMP-6 protein expression were assessed by immunocytochemistry in longitudinal femoral sections. In untreated animals, BMP-6 was expressed by a significant proportion of growth plate chondrocytes and a subpopulation of bone marrow cells. In contrast, osteoblasts were consistently BMP-6 negative. From as early as 4 days after starting estrogen, clusters of slightly elongated BMP-6-positive cells were observed within the marrow cavity; the majority were close to active bone formation surfaces. Double immunolabeling studies revealed that only approximately 10% of BMP-6-positive bone marrow cells co-expressed the osteoblast transcription factor Cbfa1 suggesting that they are largely distinct from the osteoblast precursor population generated concurrently. BMP-6-positive cells expressed neither leukocyte nor erythroid markers (CD45 and TER-119, respectively), consistent with a stromal origin. We conclude that estrogen-induced osteogenesis in female mice is associated with increased levels of BMP-6 mRNA in mouse femurs, which seems to reflect the emergence of clusters of BMP-6 positive stromal cells adjacent to active bone formation surfaces. These findings raise the possibility that BMP-6 serves as a paracrine mediator of estrogen's osteogenic action in mice.