AbstractIGF-I activates signaling pathways that increase the expression of muscle-specific genes in differentiating myoblasts. Induction of skeletal α-actin expression occurs during differentiation through unknown mechanisms. The purpose of this investigation was to examine the mechanisms that IGF-I uses to induce skeletal α-actin gene expression in C2C12 myoblasts. IGF-I increased skeletal α-actin promoter activity by 107% compared with the control condition. Ni+ [T-type voltage-gated Ca2+ channel (VGCC) inhibitor] reduced basal-induced activation of the skeletal α-actin promoter by approximately 84%, and nifedipine (L-type VGCC inhibitor) inhibited IGF-I-induced activation of the skeletal α-actin promoter by 29–48%. IGF-I failed to increase skeletal α-actin promoter activity in differentiating dysgenic (lack functional L-type VGCC) myoblasts; 30 mm K+ and 30 mm K++IGF-I increased skeletal α-actin promoter activity by 162% and 76% compared with non-IGF-I or IGF-I-only conditions, respectively. IGF-I increased calcineurin activity, which was inhibited by cyclosporine A. Further, cyclosporine A inhibited K++IGF-I-induced activation of the skeletal α-actin promoter. Constitutively active calcineurin increased skeletal α-actin promoter activity by 154% and rescued the nifedipine-induced inhibition of L-type VGCC but failed to rescue the Ni+-inhibition of T-type VGCC. IGF-I-induced nuclear factor of activated T-cells transcriptional activity was not inhibited by nifedipine or Ni+. IGF-I failed to increase serum response factor transcriptional activity; however, serum response factor activity was reduced in the presence of Ni+. These data suggest that IGF-I-induced activation of the skeletal α-actin promoter is regulated by the L-type VGCC and calcineurin but independent of nuclear factor of activated T-cell transcriptional activity as C2C12 myoblasts differentiate into myotubes.