In this study we investigated the role of extracellular 5′-guanosine-triphosphate (GTP) on early phases of skeletal muscle differentiation using the widely used C2C12 mouse cells as a myogenic model. We show that extracellular GTP binding to specific sites activates a metabotropic cascade that leads to a transient intracellular Ca2+ mobilization, consequent activation of the intermediate Ca2+-activated K+ channels (IKCa), and hyperpolarization of the plasma membrane. We further show that in differentiating C2C12 myoblasts GTP induces a proliferative boost, and increases the number of cells positive for the myosin heavy chain (MyHC) proteins. These effects were shown to be mediated by the IKCa channel-dependent hyperpolarization, as evidenced by their disappearance when myoblasts were incubated with the IKCa channel inhibitor charybdotoxin. These data give new insights into nucleotide purinergic signalling pathways, and address the role of the GTP-dependent IKCa channel activation and hyperpolarization in myogenesis.