The postmitotic nature and longevity of skeletal muscle fibers permit stable expression of any transfected gene. Direct in vivo injection of plasmid DNA, in both adult and regenerating muscles, is a safe, inexpensive, and easy approach. Here we present an optimized electroporation protocol based on the use of spatula electrodes to transfer cDNA in vivo into the adult myofibers of an anatomically defined muscle, which could be functionally characterized. In our hands, about 80% of adult myofibers were transfected in vivo by different plasmids for GFP fusion proteins or for beta-galactosidase. The luciferase activity increased several orders of magnitude when compared to standard DNA delivery. In an anatomical defined muscle, the wide gene transfer was comparable to or better than that of retrovirus delivery, that recently has been shown to be prone to severe side-effects in human clinical studies. Furthermore, with our method the tissue damage was greatly decreased. Thus, the present work describes in vivo functional electrotransfer of genes in adult skeletal muscle fibers by a protocol that is of great potential for gene therapy, as well as for basic research.