We have studied the modulation of expression and surface distribution of the molecular forms of the neural cell adhesion molecule (N-CAM) during myogenesis in vitro. We found one minor and two major N-CAM forms-180, 145 and 125 kDa respectively in primary cultures of mouse muscle cells. The 180 and 145 kDa forms were present in myoblasts before fusion. At fusion, total N-CAM increased with no 180 kDa polypeptide, but with a new 125 kDa form, together with the 145 kDa form. We determined the localization of N-CAM on the myotube surface and compared it to that of the acetylcholine receptor. N-CAM but not the receptor was found on the myoblast surface before fusion. Both proteins were uniformly distributed on the cell surface of early myotubes. Then, bright spots appeared, rapidly followed by the formation of clusters of both the acetylcholine receptor and N-CAM, at the time contractile activity was established. However these clusters were never colocalized, except after synapse formation. N-CAM clusters, but not acetylcholine receptor clusters, were dispersed following Nocodazole-induced microtubule depolymerization. We further observed that patching of N-CAM by divalent anti-N-CAM antibodies had no effect on acetylcholine receptor clusters. These results suggest that there is no mechanochemical link between the receptor and N-CAM. In myotubes, part of the 125 kDa form was released from the cell surface by the phosphatidylinositol phospholipase C. This phosphatidylinositol anchored form was mostly present outside the clusters where the 145 kDa form seems to be concentrated. Another pool of 125 kDa was insoluble in non-ionic detergent and was extracted by 0.1% SDS only. We suggest that the SDS extracted 125 kDa N-CAM is present in basal lamina. Thus, specific N-CAM forms with different interactions with basal lamina or cytoskeleton and cell surface distribution are induced during myogenesis and may be responsible for decisive modifications of cell-cell interactions involved in myoblast fusion and synaptogenesis.