It has recently been reported that tumor necrosis factor (TNF)-α has the ability to accelerate osteoclastogenesis. We previously reported that the proinflammatory cytokine interleukin (IL)-18 inhibits TNF-α-mediated osteoclastogenesis in mouse bone marrow cultures. In the present study, the effect of IL-18 on TNF-α-mediated osteoclastogenesis was investigated in vivo. We administered TNF-α with or without IL-18 into the supracalvaria of mice. The number of osteoclasts in the suture of the calvaria was increased in mice administered TNF-α. The number of osteoclasts in mice administered both TNF-α and IL-18 was lower than that in mice administered TNF-α alone. We previously showed that IL-12 and IL-18 synergistically inhibit TNF-α-mediated osteoclastogenesis in vitro. To assess the ability of these two cytokines to synergistically inhibit TNF-α-induced osteoclastogenesis in vivo, mice were administered the two cytokines at doses that did not inhibit osteoclast formation. The combination of IL-12 and IL-18 markedly inhibited TNF-α-induced osteoclastogenesis in vivo. To evaluate how IL-12 and IL-18 synergistically affect TNF-α-induced osteoclastogenesis, the IL-18 receptor (IL-18R) and IL-12R expression levels were analyzed by RT-PCR in bone marrow cells cultured with IL-12 or IL-18. IL-18R mRNA was increased in cells cultured with IL-12, while IL-12R mRNA was increased in cells cultured with IL-18. In addition, IL-18 inhibited TNF-α-induced osteoclastogenesis in mice with T-cell depletion caused by anti-CD4 and anti-CD8 antibodies. The present results suggest that IL-18 may inhibit TNF-α-mediated osteoclastogenesis in vivo via a T cell-independent mechanism.