We have established long-term human myogenic cultures from adult human skeletal muscle biopsies by infecting primary explant cultures with an amphotropic retroviral construct encoding a temperature-sensitive SV40 large T antigen, tsA58-U19. Infected myoblasts expressed the large T antigen and showed greatly enhanced proliferative capacity when cultured at 33°C, compared with noninfected cells. When the infected cultures were incubated at 39°C, the cells withdrew from cycle, aligned, and fused to form multinucleated myotubes which expressed certain antigens that are similarly expressed in nontransduced differentiating muscle cells. Myogenic clones with greatly increased proliferative capacity were generated, for the first time, from biopsies obtained from Duchenne muscular dystrophy patients as well as from normal, dystrophin-positive individuals. Cell lines produced by this approach may prove valuable forin vitrostudies of myogenesis and for investigating the cellular and molecular consequences of inherited muscle diseases.