Receptor sites, specific for guanosine 5′-triphosphate (GTP) were characterised in myoblasts and myotubes of C2C12 mouse skeletal muscle cells, using binding experiments and measurements of intracellular Ca2+ concentration ([Ca2+]i). We identified two GTP binding sites in myoblasts membranes: a high affinity site (Kd = 15.4 ± 4.6 μM; Bmax = 1.7 ± 0.5 nmol mg−1 protein); and a low affinity site (Kd = 170 ± 94.5 μM; Bmax = 14.2 ± 3.9 nmol mg−1 protein). In myotube membranes only a low affinity binding site for GTP (Kd = 169 ± 39 μM; Bmax = 12.3 ± 1.4 nmol mg−1 protein) was detected. In myoblasts GTP binding was not displaced by ATP or UTP, even at high concentrations (up to of 1 mM), but it was affected by treatments with suramin or Reactive Blue 2 (RB2), the non-selective purine receptor antagonists. In contrast, in myotubes GTP binding was partially displaced by high concentrations of ATP, but treatments with the non-selective purine receptor antagonists, suramin or RB2, and with UTP had no effect on GTP binding. The addition of GTP to myoblasts, and to myotubes, resulted in elevations of [Ca2+]i. The patterns of Ca2+ response however, were different in the two cell phenotypes. In myoblasts the addition of GTP induced two types of Ca2+ responses: (1) a fast increase in [Ca2+]i, followed by a sustained [Ca2+]i elevation, and (2) a slow raising and steady prolonged increase in [Ca2+]i. In myotubes, however only fast Ca2+ responses were observed following the addition of 500 μM GTP. In the myoblasts and myotubes GTP-stimulated [Ca2+]i increases were abolished by treatments with suramin or RB2 at concentrations which had no effect on the ATP-induced Ca2+ responses. We conclude, that C2C12 cells express two distinct binding sites for GTP before differentiation, but only one after, the low affinity binding site. These results suggest a possible role of the high affinity GTP binding site in early stage of development of skeletal muscle.