Intramuscular injection of plasmid is a potential alternative to viral vectors for the transfer of therapeutic genes into skeletal muscle fibers. The low efficiency of plasmid-based gene transfer can be enhanced by electroporation (EP) coupled with the intramuscular application of hyaluronidase. We have investigated several factors that can influence the efficiency of plasmid-based gene transfer. These factors include electrical parameters of EP, optimal use of hyaluronidase, age and strain of the host, and plasmid size. Muscles of very young and mature normal, mdx, and immunodeficient mice were injected with plasmids expressing beta-galactosidase, microdystrophin, full-length dystrophin, or full-length utrophin. Transfection efficiency, muscle fiber damage, and duration of transgene expression were analyzed. The best transfection level with the least collateral damage was attained at 175-200 V/cm. Pretreatment with hyaluronidase markedly increased transfection, which was also influenced by the plasmid size and the strain and the age of the mice. Even in immunodeficient mice, there was a significant late decline in transgene expression and plasmid DNA copies, although both still remained relatively high after 1 year. Thus, properly optimized EP-assisted plasmid-based gene transfer is a feasible, efficient, and safe method of gene replacement therapy for dystrophin deficiency of muscle but readministration may be necessary.