Our previous studies showed that a soy-protein diet prevents ovariectomy-induced bone loss. The purpose of this study was to determine whether isoflavones in soy protein are responsible for this bone-protective effect. Forty-eight 95-d-old Sprague-Dawley rats were divided into 4 groups: sham-operated fed a casein-based diet (SHAM), ovariectomized fed a casein-based diet (OVX+CASEIN), ovariectomized fed soy protein with normal isoflavone content (OVX+SOY), and ovariectomized fed soy protein with reduced isoflavone content (OVX+SOY-). The OVX+SOY group had significantly greater femoral bone density (in g/cm3 bone vol) than the OVX+CASEIN group, whereas OVX+SOY- was similar to OVX+CASEIN (mean +/- SD; SHAM, 1.522 +/- 0.041; OVX+CASEIN, 1.449 +/- 0.044; OVX+SOY, 1.497 +/- 0.030; OVX+SOY-, 1.452 +/- 0.030). Ovariectomy resulted in greater bone turnover as indicated by higher serum alkaline phosphatase activity, serum insulin-like growth factor I and insulin-like growth factor binding protein 3 concentrations, and urinary hydroxyproline. These increases were not affected by soy with either normal or reduced isoflavone content. Similarly, histomorphometry revealed a greater bone formation rate with ovariectomy, and this was not altered by the soy diets. The findings of this study suggest that isoflavones in soy protein are responsible for its bone-sparing effects. Further studies to evaluate the mechanism of action of isoflavones on bone are warranted.