Skeletal muscle cells are continuously exposed to oxidative stress. Thus, they compensate environmental challenges by increasing adaptive responses, characterized by activating protein 1 (AP-1)- and nuclear factor kappaB (NF-kappaB)-mediated transcriptional upregulation of endogenous enzymatic and nonenzymatic antioxidants. We investigated the crosstalk of molecules involved in redox signaling in muscle cells, by using the rat L6C5 and mouse C2C12 cell lines, which represent a useful experimental model for studying muscle metabolism. We analyzed specific antioxidant systems, including glutathione, thioredoxin reductase, and antioxidant enzymes, and the redox-sensitive transcription factors AP-1 and NF-kappaB, in both myoblasts and myotubes. We found that the high levels of NF-kappaB DNA binding activity and thioredoxin reductase, together with inhibitory AP-1 complexes, allowed increased expression of antioxidant enzymes and survival of C2C12 cells after oxidant exposure. On the contrary, L6C5 myoblasts had a sensitive phenotype, correlated with lower levels of thioredoxin reductase, catalase, and NF-kappaB activity and higher levels of GSSG and activating AP-1 complexes. Interestingly, this cell line acquired an apoptosis-resistant phenotype, accompanied by drastic changes in the oxidant/antioxidant balance, when induced to differentiate. In conclusion, the two cell lines, although similar in terms of growth and differentiation, displayed significant heterogeneity in terms of redox homeostasis.