Contracting skeletal muscle produces and releases interleukin-6 (IL-6) in high amounts. Nevertheless, the mechanisms underlying IL-6 expression are not understood. Since inositol-1,4,5-trisphosphate (IP₃) mediated slow Ca ⁺² signals evoked by depolarization of skeletal myotubes appear to play a role in the regulation of gene expression, we examined its involvement on IL-6 transcription. Using semi-quantitative RT-PCR, we have shown that K⁺-depolarization of myotubes induces a transient increase in IL-6 mRNA level which peaks at 3-4 h and is independent of extracellular Ca⁺². Inhibitors of IP₃-dependent Ca⁺² signals, like 2-aminoethoxydiphenyl borate (2-APB) and U73122, decreased activation of IL-6 gene expression as did Ca⁺² signals inhibitor BAPTA-AM whereas ryanodine, a fast Ca⁺² transient inhibitor, had no effect on IL-6 induction. Depolarization of myotubes transiently transfected with a reporter gene construct, containing 651 bp of IL-6 promoter, induced a two fold increase on promoter activity, which was abolished by either 2-APB or U73122 and remained unaffected after ryanodine treatment. Site-directed mutagenesis of parental construct allowed us to identify AP-1 and NF-B sequences as regulatory elements involved in IL-6 up-regulation. Our results provide evidence for involvement of IP₃-mediated Ca⁺² signals on IL-6 transcription in skeletal muscle cells.