We demonstrate that self-renewing myoblasts can be identified in the progeny of single human muscle satellite cells (HMSC) in culture. We show, using cytoskeletal proteins and cell size as markers, that self-renewing myoblasts are phenotypically different from other myoblasts, but similar to native HMSC. Native desmin-positive HMSC, cultured as single cells, yielded two major populations of myoblasts, alpha-sarcomeric (alpha-SR)-actin-positive myoblasts and desmin-positive myoblasts. In appropriate culture conditions, alpha-SR-actin-positive myoblasts fused into myotubes, whereas a population of desmin-positive non-fusing myoblasts (NFMB) persisted for weeks among the myotubes. Upon isolation from myotubes, some of the NFMB resumed proliferation and their progeny included fusing and non-fusing myoblasts, with the same cytoskeletal phenotypes as the progeny of native HMSC. This self-renewal cycle could be repeated, yielding four cohorts of myoblasts. The yield of self-renewing cells appeared to decrease with the number of cycles. These results suggest that stem cells are present among NFMB. Moreover, we find that these presumptive stem cells are already segregated during myoblast proliferation. They are small, phenotypically similar to native HMSC, and do not divide unless they are isolated from their sister progeny and cultured alone. Enriched preparations of cells with stem cell-like properties can be obtained from proliferating myoblasts by flow cytometry on the basis of size and nucleocytoplasmic ratio.