Myoblast transplantation has been investigated as a therapy for muscle-related diseases and as a gene delivery vehicle for therapeutic recombinant proteins. Clinical successes involving muscle cell transplantation have been limited, in part because of poor donor cell survival, and the heterogeneous nature of myogenic donor cells has largely been ignored. We have previously reported an isolation technique, preplating, that results in purified myogenic cells that are capable of significantly higher rates of donor cell survival leading to enhanced gene transfer to skeletal muscle. Characterization of these purified cells revealed that they display markers common to stem cells and are capable of multilineage differentiation. This study was performed to phenotypically characterize, by flow cytometry, muscle-derived cell populations obtained by the preplate technique for the purpose of eventually developing a method to quickly identify and isolate viable muscle cells best suited for transplantation. Muscle cell cultures were analyzed for expression of the surface proteins Sca-1, c-Kit, and CD34. We found that the preplate technique purifies distinct myogenic cell subpopulations expressing CD34 alone (Sca-1 negative) and Sca-1 alone (CD34 negative), but that this expression is subject to change with time in culture. Isolation and transplantation of phenotypically pure Sca-1-positive myogenic cells, obtained by magnetic cell sorting, demonstrates the ability to quickly select viable myogenic cells capable of regenerating skeletal muscle and restoring dystrophin expression within dystrophic host skeletal muscle. Flow cytometric described phenotypes will aid in the rapid isolation of specific donor cell populations for muscle cell transplants and muscle cell-mediated gene therapies, thereby enhancing their future success.