We have developed an in vitro system for the study of postnatal human muscle under standardized conditions. The technique utilizes cloning to isolate pure populations of muscle cells. By manipulating culture conditions we can maximize either proliferation or differentiation of individual clones or of clones pooled to yield mass cultures of muscle cells. The muscle phenotype is stable; cells can be stored in liquid nitrogen for long-term use without loss of proliferative or differentiative potential. We have determined proliferative capacity of muscle cells from an analysis of clonal growth kinetics; we determined differentiative capacity from morphological evidence (cell fusion, striations, contractions, and the appearance of acetylcholine receptors) and biochemical analysis of muscle protein synthesis (creatine kinase, alpha-actin, tropomyosin, and myosin light chains). Our approach eliminates the variability in cellular composition that has complicated studies of primary muscle to date. We can now study in a controlled fashion the interactions and contributions of different cell types to the development of normal and genetically dystrophic human muscle.