Considerable evidence supports the hypothesis that estrogen prevents bone loss by blocking the production of cytokines in bone or bone marrow. However, controversy remains on the role of candidate factors, such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor (TNF). As IL-1 and TNF have many additive and/or synergistic effects in bone, we tested the hypothesis that the simultaneous block of IL-1 and TNF is required to prevent the initial phase of rapid bone loss that follows ovariectomy (ovx). To this aim, rats were ovariectomized and treated for 2 weeks with either IL-1 receptor antagonist (IL-1ra), an inhibitor of IL-1, or TNF-binding protein (TNFbp), an inhibitor of TNF. Ovx increased bone marrow cell secretion of IL-1 and TNF and decreased the bone density of the distal femur, as measured by dual energy x-ray absorptiometry. Ovx-induced bone loss was decreased by both IL-1ra and TNFbp and completely prevented by simultaneous treatment with IL-1ra and TNFbp. Combined treatment with IL-1ra and TNFbp decreased urinary pyridinoline cross-links, a marker of bone resorption that reflects osteoclast number and osteoclast activity, whereas treatment with either inhibitor alone was less effective. Both IL-1ra and TNFbp decreased the number of osteoclasts on the endocortical surfaces and stimulated bone formation, but the two inhibitors had no additive effects on these indexes, suggesting that inhibition of osteoclastogenesis and stimulation of bone formation do not account for the additive bone-sparing effects of IL-1ra and TNFbp. These inhibitors had no effect in sham-operated rats, indicating that they specifically blocked estrogen-dependent events. In conclusion, these data indicate that in the early post-ovx period, IL-1 and TNF play a critical causal role in inducing bone loss and do so by stimulating bone resorption and inhibiting bone formation.