Gene transfer into the peritoneal cavity by nonviral methods may provide an effective therapeutic approach for peritoneal diseases. Herein, we investigated the feasibility and the effectiveness of ultrasound-microbubble-mediated delivery of naked plasmid DNA into the peritoneal cavity in rats. Following the intraperitoneal or the intravenous administration of a mixture of plasmid DNA (100 microg) and ultrasound contrast agent microbubbles, an ultrasound transducer was applied on the abdominal wall. The reporter pTRE plasmid encoding Smad7 was used to evaluate transfection efficiency. Smad7 expression was induced by doxycycline in drinking water. We detected less than 10% apoptotic cells and no inflammatory reaction in peritoneal tissues following the ultrasound-microbubble-mediated transfection. More importantly, the insonation significantly improved the transfection efficiency in peritoneal tissues. The transfection efficiency by intraperitoneal delivery route was higher than the intravenous route. The reporter gene, pTRE-Smad7, was readily detected in the parietal peritoneum, mesentery, greater omentum and adipose tissue. The peak of transgene expression occurred 2 days after transfection and the transgene expression diminished in a time-dependent manner thereafter. Overall, the effectiveness and simplicity of the ultrasound-microbubble-mediated system may provide a promising nonviral means for improving gene delivery for treating peritoneal diseases in vivo.