<jats:title>Abstract</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>The use of ultrasound (US)‐facilitated gene therapy is increasing rapidly as a result of its high specificity and non‐invasiveness. However, the acoustic parameters that produce the most efficient transfection have not been established. The present study investigated the effects of time parameters [including pulsing strategy (on‐ and off‐times), exposure duration, pore opening time and expression duration] of US‐facilitated gene transfection.</jats:p></jats:sec><jats:sec><jats:title>Methods</jats:title><jats:p>Cervical cancer cells (HeLa cells) cultured with pCMViLUC plasmids were exposed to 1‐MHz pulsed US, and gene transfection efficiency and cell viability were assessed. The ability of <jats:italic>in vivo</jats:italic> transfection by ultrasound using different pulsing strategy was also evaluated.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>For a constant total exposure time, the transfection was always better for a longer on‐time (on–off ratio = 1, or 50% duty cycle) than a shorter one (ratio = 0.1, 9% duty cycle), whether performed <jats:italic>in vitro</jats:italic> or <jats:italic>in vivo</jats:italic>. However, for a fixed on‐time, there was a strong inverse correlation between transfection efficiency and off‐time (<jats:italic>r</jats:italic><jats:sup>2</jats:sup> = 0.89). Multiple regression analysis showed that the on‐time, off‐time and on–off ratio are all independent variables for US‐facilitated gene transfection. Furthermore, transfection increased with the total exposure time, but plateaued for long exposure times. More than half of the opened pores closed within 15 s after the cessation of US exposure. The half‐life of the transfected DNA was 7.43 days.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>Time is an important determinant of US‐facilitated gene transfection. Methods for increasing the transfected number of plasmids, such as increasing the concentration of plasmids or the duration of pore opening, will be critical for future research and clinical applications. Copyright © 2009 John Wiley & Sons, Ltd.</jats:p></jats:sec>