Cellular therapy of Duchenne muscular dystrophy is currently showing encouraging results. However, there is still no serum-free medium that allows multiplying functional myoblasts, the cells required for this therapy, obtained from healthy donors, in sufficient number. A systematic screen for a defined culture medium that would allow expanding fully functional human myoblasts at the same rate and to the same extent as the existing serum-containing medium has been undertaken. To this end, 6 basal media and more than 60 defined additives have been screened for their individual as well as synergistic effects. The medium developed in this study permits a 12-log expansion of human myoblasts over a 60-day period. The cells obtained have the same capacity to fuse and form myofibres as myoblasts multiplied in serum-containing medium.