We developed a muscle-specific gene delivery system based on two-step gene transfer. The first step involved adenovirus-mediated transfer of the ecotropic retrovirus receptor (EcoRec) gene driven by the muscle-specific desmin promoter. Both human primary myoblasts and fibroblasts were efficiently transduced with this adenovirus vector. However, expression of EcoRec was detected only in myoblasts, In the second step, EcoRec-expressing myoblasts could be stably transduced with the ecotropic retroviral vector with the P-galactosidase gene. Approximately 15% of myoblasts were transduced by this two-step strategy, When the transduced myoblasts were differentiated into myotubes, extensive cell-cell fusion occurred, and the apparent number of P-galactosidase-positive cells increased to 28%, These results indicate that our two-step gene delivery system could be used for targeted and stable gene transfer into muscle cells.