Muscle wasting is often associated with chronic inflammation. Because tumor necrosis factor alpha (TNF-alpha) has been implicated as a major mediator of cachexia, its effects on C2C12 myocytes were examined. TNF-alpha activated nuclear factor-kappaB (NF-kappaB) and interfered with the expression of muscle proteins in differentiating myoblasts. Introduction of a mutant form of inhibitory protein kappaBalpha (IkappaBalpha) restored myogenic differentiation in myoblasts treated with TNF-alpha or interleukin 1beta. Conversely, activation of NF-kappaB by overexpression of IkappaB kinase was sufficient to block myogenesis, illustrating the causal link between NF-kappaB activation and inhibition of myogenic differentiation. The inhibitory effects of TNF-alpha on myogenic differentiation were reversible, indicating that the effects of the cytokine were not due to nonspecific toxicity. Treatment of differentiated myotubes with TNF-alpha did not result in a striking loss of muscle-specific proteins, which shows that myogenesis was selectively affected in the myoblast stage by TNF-alpha. An important finding was that NF-kappaB was activated to the same extent in differentiating and differentiated cells, illustrating that once myocytes have differentiated they become refractory to the effects of NF-kappaB activation. These results demonstrate that inflammatory cytokines may contribute to muscle wasting through the inhibition of myogenic differentiation via a NF-kappaB-dependent pathway.