The calcium-dependent fusion of cultured rat myoblasts to multinucleate myotubes appears to require the activity of a neutral metalloendoprotease at the time of fusion. Metalloendoprotease inhibitors and synthetic dipeptide substrates prevent myoblast fusion when added to fusion-competent myoblasts with the addition of calcium. Metalloendoprotease activity has been identified and partially characterized in myoblast membranes with a fluorogenic protease substrate, and is inhibited by the same compounds that prevent fusion.