This study is an investigation to experimentally confirm that ultrasound (US) irradiation is effective in enhancing the gene expression of plasmid DNA. A cationized gelatin was prepared by introducing primary amino groups into gelatin. The plasmid of the LacZ gene was complexed with the cationized gelatin and injected into the femoral muscle of normal mice. Following US irradiation at the injected site of muscle, the gene expression of the treated muscle was evaluated to compare with that of the complex injection plus no US irradiation. US irradiation enabled the complex to significantly enhance the gene expression at the injected muscle, in contrast to naked plasmid DNA, although the extent depended on the experimental conditions, such as the injection dose of plasmid DNA, and the time period or timing of US irradiation. The gene-expressed area at the muscle was not changed by the time interval between the complex injection and US irradiation. Fluorescent microscopic observation revealed that the complex was homogeneously distributed in the muscle tissue around the injected site by US irradiation without any dissociation of plasmid DNA. The fluorescent image of plasmid DNA superposed on that of myosin heavy chain indicated intracellular localization of plasmid DNA. We demonstrated that US irradiation is a promising technique to enhance gene expression even in the normal muscle.