Osteoclast-like cells, in cocultures with mouse spleen cells and clonal osteogenic stromal ST2 cells, are formed from spleen cells with monocyte/macrophage lineage in response to a combination of osteoclast differentiation factor (RANKL) and OPG, a decoy receptor for RANKL, produced by ST2 cells in response to 1alpha,25-dihydroxyvitamin D(3). Treatment of ST2 cells with the natural isoflavonoid genistein for 6 h before coculture with spleen cells inhibited the formation of tartrate-resistant acid phosphatase-positive osteoclast-like cells. When we measured levels of RANKL mRNA in ST2 cells, we found that genistein decreased the level of this mRNA. By contrast, the level of OPG mRNA was enhanced by genistein. Genistein is a specific inhibitor of topoisomerase II (topo II) and an inhibitor of protein tyrosine kinase, as well as being a potent phytoestrogen. To characterize the mode of action of genistein, we examined the effects of an inactive form of genistein (daidzein), 17beta-estradiol, inhibitors of topo II, and inhibitors of tyrosine kinases on the formation of tartrate-resistant acid phosphatase-positive osteoclast-like cells. Among the compounds tested, two inhibitors of topo II, amsacrine and etoposide, attenuated the formation of osteoclast-like cells via reciprocal regulation of the expression of mRNAs for RANKL and OPG in ST2 cells, acting similarly to genistein. Our findings indicate that genistein might inhibit the formation of osteoclast-like cells via inhibition of the activity of topo II, suggesting the novel possibility that topo II might play an important role in osteoclastogenesis.