During in vitro myogenesis, mononucleated myoblasts fuse among themselves to form multinucleated myotubes. We have recently reported for the first time in the literature that a Ca2(+)-dependent ectoprotein kinase is responsible for this process, but we had no direct evidence for the role of extracellular ATP. To investigate whether the cells can fuse or not in the absence of this nucleotide, we used a nucleotide affinity label, fluorosulfonylbenzoyl adenosine (FSBA). We report here its use in detecting the nucleotide-binding sites at the cell surface of intact myoblasts in culture. We demonstrate that FSBA blocks fusion by inhibiting the ectoprotein kinase activity of the cells at sublethal concentrations. Radioactive [14C]SBA is incorporated into seven cell surface proteins and into the 48-kDa protein, among others. This species is specific for fusion-competent myoblasts and is implicated in this process. This is the first time that nucleotide-binding molecular species have been identified at the surface of myoblasts.