Tumor necrosis factor-alpha (TNF-alpha) is a pleiotropic cytokine produced by activated macrophages. Nitric oxide (NO) is a highly reactive nitrogen radical implicated in inflammatory responses. We investigated the signaling pathway involved in inducible nitric oxide synthase (iNOS) expression and NO production stimulated by TNF-alpha in cultured myoblasts. TNF-alpha stimulation caused iNOS expression and NO production in myoblasts (G7 cells). TNF-alpha-mediated iNOS expression was attenuated by integrin-linked kinase (ILK) inhibitor (KP392) and siRNA. Pretreatment with Akt inhibitor, mammalian target of rapamycin (mTOR) inhibitor (rapamycin), NF-kappaB inhibitor (PDTC), and IkappaB protease inhibitor (TPCK) also inhibited the potentiating action of TNF-alpha. Stimulation of cells with TNF-alpha increased ILK kinase activity. TNF-alpha also increased the Akt and mTOR phosphorylation. TNF-alpha mediated an increase of NF-kappaB-specific DNA-protein complex formation, p65 translocation into nucleus, NF-kappaB-luciferase activity was inhibited by KP392, Akt inhibitor, and rapamycin. Our results suggest that TNF-alpha increased iNOS expression and NO production in myoblasts via the ILK/Akt/mTOR and NF-kappaB signaling pathway.