Tumor necrosis factor-alpha (TNF-alpha) stimulates osteoblast production of interleukin-6 (IL-6), an inflammatory cytokine implicated in osteoclastic bone resorption. Therefore, we tested the hypothesis that TNF-alpha-induced IL-6 production in MG-63 osteosarcoma cells occurs via the p38 mitogen-activated protein kinase (MAPK) pathway. TNF-alpha activated p38 MAPK and stimulated IL-6 secretion by MG-63 cells, and pre-incubation of cells with the p38 MAPK inhibitor abrogated TNF-alpha-dependent IL-6 secretion. Transfection of IL-6 full-length and 5-deletion gene promoter reporter constructs indicated that p38 MAPK activation by TNF-alpha enhanced IL-6 gene expression, and that the p38 MAPK-responsive region resided in the proximal 260-bp segment. Transfection of NFkappaB and C/EBPbeta-sensitive reporter promoter constructs demonstrated that NFkappaB activity was enhanced and that constitutive C/EBPbeta was inhibited by TNF-alpha, with both effects being p38 MAPK-dependent. In conclusion, although p38 MAPK activation by TNF-alpha stimulates IL-6 secretion by MG-63 cells, it has opposing effects on c/EBPbeta and NFkappaB activity.