In the present study we have tested the ability of reactive oxygen species (ROS) to stimulate the production of interleukin (IL)- 6 from skeletal myocytes. Differentiated C2C12 murine skeletal muscle cells (myotubes) exposed to pyrogallol (PYR), xanthine/ xanthine-oxidase (X/XO), or H₂O₂ for 24 h exhibited a concentration-dependent increase in IL-6 production. Unlike myotubes, incubation of myoblasts and endothelial cells with X/XO or PYR did not result in increased IL-6 release. In myotubes, superoxide dismutase and catalase blocked the ROS-induced IL-6 release. Exposure of myotubes to H₂O₂ increased steady-state IL-6 mRNA levels, and pretreatment of myotubes with actinomycin D or cycloheximide abolished the ROS-induced IL-6 production. In addition, pretreatment of cells with N-acetyl-cysteine blocked tumor necrosis factor (TNF)- α –induced IL-6 release, suggesting that endogenously produced ROS participate in IL-6 production. Myotubes stimulated with H₂O₂ exhibited increased I κ B- α phosphorylation and degradation, and treatment of C2C12 with ROS-generating agents increased activator protein (AP)-1 and nuclear factor (NF)- κ B–dependent promoter activity. Finally, preincubation of myotubes with the pharmacologic inhibitor of NF- κ B, diethyldithiocarbamate, or transient transfection with an I κ B- α mutant, inhibited the ROS-stimulated IL-6 release. In conclusion, ROS stimulate IL-6 production from skeletal myotubes in a manner that involves transcriptional activation of the IL-6 gene through an NF- κ B–dependent pathway.