Sphingosine, a potent inhibitor of protein kinase C, was found to block membrane fusion of chick embryonic myoblasts in culture. This effect was dose-dependent and could be reversed upon removal of the drug. Treatment with 12-O-tetradecanoylphorbol 13-acetate, which is a powerful activator of protein kinase C and capable of preventing myoblast fusion, further potentiated the inhibitory effect of sphingosine. Thus, the sphingosine-mediated inhibition of myoblast fusion appears to be independent of protein kinase C. Sphingosine also decreased the phosphorylation state of the 100-kDa protein when given to the cell extracts, and this inhibition was competitive with calmodulin. Thus, sphingosine seems to act as a calmodulin antagonist. These results suggest that the sphingosine-mediated inhibition of myoblast fusion may be associated with the inhibitory effect of the drug against the calmodulin-dependent phosphorylation of the 100-kDa protein.