Myogenesis is characterized by membrane fusion and accumulation of muscle specific proteins. We have previously shown that nitric oxide acts as a messenger for membrane fusion. Here we show that inhibitors of the proteasome, such as lactacystin, reversibly block both the fusion of L6 myoblasts and the accumulation of muscle specific proteins, such as myosin heavy chain (MHC). The inhibitors also reversibly prevented the induction of the NF-kappaB activity, which is required for the expression of nitric oxide synthase (NOS). Moreover, the inhibition of the NF-kappaB activity occurred in parallel with that of the NOS activity upon treatment with increasing concentrations of lactacystin. While pyrrolidine dithiocarbamate, an inhibitor of NF-kappaB, blocked both membrane fusion and accumulation of MHC, N(G)-monomethyl-L-arginine, a specific inhibitor of NOS, inhibited only the fusion. These results suggest that the proteasome plays an essential role in the regulation of myogenic differentiation through the activation of NF-kappaB and that the target of NF-kappaB for the expression of muscle specific proteins is distinct from that for myoblast fusion.