Contracting skeletal muscle produces and releases interleukin-6 (IL-6) in high amounts. Nevertheless, the mechanisms underlying IL-6 expression are not understood. Because inositol-1,4,5-trisphosphate (IP(3))-mediated slow Ca(2+) signals evoked by depolarization of skeletal myotubes appears to play a role in the regulation of gene expression, we examined its involvement on IL-6 transcription. With the use of semiquantitative 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 IP(3)-dependent Ca(2+) signals, like 2-aminoethoxydiphenyl borate (2-APB) and U-73122, 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 twofold increase in promoter activity, which was abolished by either 2-APB or U-73122 and remained unaffected after ryanodine treatment. Site-directed mutagenesis of parental construct allowed us to identify activator protein-1 and NF-kappaB sequences as regulatory elements involved in IL-6 upregulation. Our results provide evidence for involvement of IP(3)-mediated Ca(2+) signals on IL-6 transcription in skeletal muscle cells.