Tumor necrosis factor (TNF) induces osteoclast differentiation from bone marrow cells in the presence of macrophage colony-stimulating factor. Treatment of bone marrow cells with SB203580 but not PD98059 inhibited TNF-induced osteoclast differentiation. In RAW264 cells which differentiate into osteoclast-like multinucleated cells by TNF treatment alone, activation of p38 mitogen-activated protein (MAP) kinase induced by murine TNF was comparable to and independent of the receptor activator of necrosis factor-κB ligand. Moreover, the number of multinucleated osteoclasts induced by TNF in bone marrow cell cultures derived from p38 MAP kinase gene deficient mice was significantly less than that from control mice. These results indicate that the p38 MAP kinase pathway plays a crucial role in TNF-mediated osteoclast differentiation.