Although estrogen is important in human skeletal homeostasis, the major target cell in bone is unknown. Estrogen receptors (ER) have been demonstrated in osteoblasts and bone marrow stromal cells, but their presence in osteoclasts remains controversial because completely pure preparations have not been available. We have examined expression of ER-α and ER-β messenger RNA (mRNA) by RT-PCR in samples from human giant cell tumor of bone (GCT), including: whole tumor, cultured mononuclear cells, and a pure osteoclast population obtained by microisolation. Whole tumor expressed both ER-α and calcitonin receptor (CTR) mRNA and apparently lower levels of ER- β mRNA. Passaged cultures of tumor mononuclear stromal cells also expressed ER-α and low ER-β but not CTR mRNA. In pure preparations of microisolated osteoclasts, expression of ER-α or ER-β mRNA was not detected, whereas expression of CTR mRNA was readily identified. Microisolated GCT mononuclear cells expressed ER-α, but no detectable CTR mRNA. Fluorescence in situ hybridization (FISH) using an ER-α riboprobe demonstrated strong signal in the mononuclear cells but multinucleated osteoclasts showed no detectable signal. In contrast, CTR mRNA was detected in multinucleated osteoclasts but not in stromal-like tumor cells by FISH. 17β-estradiol consistently showed no effect on bone resorbing activity of osteoclasts from GCT cultured on cortical bone, although calcitonin was a potent inhibitor. These findings indicate that significant expression of ER does not occur in osteoclasts derived from human GCT and suggest that estrogen effects are mediated by other cells of the bone environment.