Gene electrotransfer is an effective nonviral technique for delivery of plasmid DNA into tissues. From a clinical perspective, muscle is an attractive target tissue as long-term, high-level transgenic expression can be achieved. Spatial distribution of the transgenic protein following gene electrotransfer to muscle in a large animal model has not yet been investigated. In this study, 17 different doses of plasmid DNA (1-1500 μg firefly luciferase pCMV-Luc) were delivered in vivo to porcine gluteal muscle using electroporation. Forty-eight hours post treatment several biopsies were obtained from each transfection site in order to examine the spatial distribution of the transgenic product. We found a significantly higher luciferase activity in biopsies from the center of the transfection site compared to biopsies taken adjacent to the center, 1 and 2 cm along muscle fiber orientation (p<0.05 and p<0.0001, respectively). On average, 43% of the total luciferase activity was localized in the center biopsy. In conclusion, we found that gene electrotransfer to muscle in a large animal model led to localized gene expression corresponding to the area delineated by the electrodes. High doses of plasmid DNA did not lead to a larger area of the muscle expressing the transgenic protein.