The deficiency of dystrophin, a sarcolemmal associated protein, is responsible for Duchenne muscular dystrophy (DMD). Gene replacement is attractive as a potential therapy. In this article, we describe a new method for myoblast transplantation and expression of dystrophin in skeletal muscle tissue of dystrophin-deficient mdx mouse through iliac vessels extracorporeal circulation. We evaluated the extracorporeal circulation as an alternative route of delivering myoblasts to the target tissue. Two series of experiments were performed with the extracorporeal circulation. In a first series, L6 rat myoblasts, transfected with LacZ reporter gene, were perfused in limbs of 15 rats. In the second series, the muscle limbs of three 6–8-week-old mdx were perfused with myoblasts of donor C57BL10J mice. Before these perfusions, the right tibialis anterior (TA) muscle of the rats and mdx was injected three times at several sites with bupivacaine (BPVC) and hyaluronidase. The ability of injected cells to migrate in the host tissue was assessed in rats by technetium-99m cell labeling. No radioactivity was detected in the lungs, bowels, and liver of animals treated with extracorporeal circulation. The tissue integration and viability of the myoblasts were ultimately confirmed by genetic and histochemical analysis of LacZ reporter gene. Following a single extracorporeal perfusion of myoblasts from donor C57BL10J, sarcolemmal expression of dystrophin was observed in clusters of myofibers in tibialis anterior muscles previously treated with BPVC and hyaluronidase. Furthermore, large clusters of dystrophin-positive fibers were observed in muscles up to 21 days after repeated treatments. These clusters represented an average of 4.2% of the total muscle fibers. These results demonstrate that the extracorporeal circulation allows selective myoblast-mediated gene transfer to muscles, opening new perspectives in muscular dystrophy gene therapy.