Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic cytokine produced by activated macrophages. IL-6 is a multifunctional cytokine that plays a central role in both innate and acquired immune responses. We investigated the signaling pathway involved in IL-6 production stimulated by TNF-alpha in cultured myoblasts. TNF-alpha caused concentration-dependent increases in IL-6 production. TNF-alpha-mediated IL-6 production was attenuated by focal adhesion kinase (FAK) mutant and siRNA. Pretreatment with phosphatidylinositol 3-kinase inhibitor (PI3K; Ly294002 and wortmannin), Akt inhibitor, NF-kappaB inhibitor (pyrrolidine dithiocarbamate, PDTC), and IkappaB protease inhibitor (L-1-tosylamido-2-phenyl phenylethyl chloromethyl ketone, TPCK) also inhibited the potentiating action of TNF-alpha. TNF-alpha increased the FAK, PI3K, and Akt phosphorylation. Stimulation of myoblasts with TNF-alpha activated IkappaB kinase alpha/beta (IKKalpha/beta), IkappaBalpha phosphorylation, p65 phosphorylation, and kappaB-luciferase activity. TNF-alpha mediated an increase of kappaB-luciferase activity which was inhibited by Ly294002, wortmannin, Akt inhibitor, PDTC and TPCK or FAK, PI3K, and Akt mutant. Our results suggest that TNF-alpha increased IL-6 production in myoblasts via the FAK/PI3K/Akt and NF-kappaB signaling pathway.