We examined the effects of human recombinant tumor necrosis factor-α (TNF) on human primary myoblasts. When added to proliferating myoblasts, TNF inhibited the expression of α-cardiac actin, a muscle-specific gene whose expression is observed at low levels in human myoblasts. TNF also inhibited muscle differentiation as measured by several parameters, including cell fusion and the expression of other muscle-specific genes, such as α-skeletal actin and myosin heavy chain. Muscle cells were sensitive to TNF in a narrow temporal window of differentiation. Northern (RNA) blot and immunofluorescence analyses revealed that human muscle gene expression became unresponsive to TNF coincident with myoblast differentiation. When TNF was added to differentiated myotubes, there was no effect on muscle gene expression. In contrast, TNF-inducible mRNAs such as interferon β-2 still responded, suggesting that the signal mediated by TNF binding to its receptor had no effect on muscle-specific genes after differentiation.