The effect of genistein on osteoclast-like multinucleated cells (MNCs) from rat femoral tissues was investigated. When rat marrow cells were cultured for 7 d in a medium containing either vehicle, parathyroid hormone (10(-8) M) or prostaglandine E2 (10(-6) M), the formation of tartrate-resistant acid phosphatase (TRACP)-positive MNCs was significantly increased. This increase was markedly weakened by the presence of genistein (10(-6) or 10(-5) M). The bone cells isolated from rat femoral tissues were cultured for 48 h in an alpha-minimal essential medium (5% fetal bovine serum) containing either vehicle or genistein (10(-8) to 10(-5) M). Osteoclasts were estimated by staining for TRACP, a marker enzyme of osteoclasts. The presence of genistein caused a significant decrease in the number of osteoclasts. Such a decrease was also seen in the presence of calcitonin (10(-10) to 10(-8) M), dibutyryl cyclic adenosine 5'-monophosphate (DcAMP; 10(-6) and 10(-5) M), calcium chloride (10(-4) and 10(-3) M) or daidzein (10(-7) to 10(-5) M). The suppressive effect of genistein (10(-5) M) was not further enhanced in the presence of calcitonin (10(-8) M), DcAMP (10(-5) M), or calcium chloride (10(-3) M), and was completely abolished by the presence of dibucaine (10(-6) M) or staurosporine (10(-7) M), both of which are inhibitors of Ca2+-dependent protein kinases. Ca2+ ionophore (A23187; 10(-6) M) induced a remarkable decrease in the number of osteoclasts in the absence or presence of genistein (10(-5) M) or calcium chloride (10(-3) M). The present study demonstrates that genistein has a direct suppressive effect on osteoclasts in vitro, suggesting that the isoflavone may induce apoptosis which is mediated through the pathway of intracellular Ca2+ signaling.