Increased transgene expression after plasmid transfer to the skeletal muscle is obtained with electroporation in many species, but optimal conditions for individual species and muscle group are not well defined. Using a muscle-specific plasmid driving the expression of a secreted embryonic alkaline phosphatase (SEAP) reporter gene, we have optimized the electroporation conditions in a large mammal model, i.e. pig. The parameters optimized include electric field intensity, number of pulses, lag time between plasmid injection and electroporation, and plasmid delivery volume. Constant current pulses, between 0.4 and 0.6 A, applied 80 s after the injection of 0.5 mg SEAP-expressing plasmid in a total formulation volume of 2 mL produced the highest expression in semimembranosus muscle in pigs. These results could be extrapolated for a different muscle group in pigs, the biceps femoris, and may be an evaluation starting point for large muscle in veterinary species or humans (see Note 1 ).