Background: Ultrasound (US) has been widely used to improve the efficiency of nonviral vector transfection. The mechanism of plasmid uptake is usually attributed to sonoporation, although there is not clear evidence for this attribution. Based on our previous results, we hypothesized that other mechanisms, such as endocytosis, could be involved in this process. Methods: NIH3T3 cells were transfected with plasmid vector pEGFP-N3 (4.7kb) using a therapeutic US without microbubbles. Bioeffects such as calcium influx, reactive oxygen species (ROS) generation and membrane potential alterations were accessed with fluorescent dyes in real-time by confocal microscopy after US insonation. Localization of labeled plasmid DNA in cells was also monitored with endocytosis markers using an immunofluorescence assay. Results: US at 2W/cm 2 with a duty-cycle of 20% for 30s resulted in approximately 40% transfection efficiency but, at 1W/cm 2, resulted in a very low level of transfection. Both the production of ROS and calcium influx were augmented during the insonation, although they were stopped soon after turning off US, with the exception of calcium influx with 1W/cm 2. US also changed the cell membrane potential to the hyperpolarization state, which returned to the normal state soon after insonation. Labeled plasmids DNA could be co-localized with clathrin-mediated endocytosis marker but not with caveolin-1. Conclusions: The present data indicate that plasmid DNA uptake promoted by US should occur via clathrin-mediated endocytosis.© 2011 John Wiley & Sons, Ltd.