Interest in the potential role of ultrasound for non-diagnostic applications such as wound healing and drug delivery has been growing rapidly. Development of such non-diagnostic technologies will require a fundamental understanding of interactions between the acoustic wave and phospholipid membranes, be they cell membranes or liposome bilayers. This work investigates the effect on membrane permeation (leakage) in vitro during exposure to ultrasound applied in two frequency ranges: “conventional” (1 MHz and 1.6 MHz) therapeutic ultrasound and low (20 kHz) frequency ultrasound. The membrane properties were modified by changes in vesicles; egg phosphatidylcholine vesicles with sizes ranging from 100 nm to 1 ěm were prepared. Leakage was quantified in terms of temporal fluorescence intensity changes observed during carefully controlled ultrasound ON/OFF time intervals. Custom-built transducers operating at frequencies of 1.6 MHz (focused) and 1.0 MHz (unfocused) were used, the Ispta of which were 46.9 W/cm2 and 3.0 W/cm2, respectively. A commercial 20 kHz, point-source, continuous wave transducer with an Ispta of 0.13 W/cm2was also used for comparative purposes. Whereas complete leakage was attained for all vesicle sizes at 20 kHz, no leakage was observed from vesicles smaller than100 nm diameter at 1.6 or 1.0 MHz. However, introducing leakage at the higher frequencies became feasible when larger (greater than 300 nm) vesicles were used, and the extent of leakage correlated well with vesicle sizes between 100 nm and 1 µm. This observation suggests that membrane properties play a crucial role in ultrasound mediated membrane permeation and that low frequency (tens of kilohertz) ultrasound exposure is more effective in introducing permeability change than the “conventional” (1 MHz) therapeutic frequency. The results of this work might help to optimize acoustic field and membrane parameters for gene or drug delivery. The outcome of this work might also be useful in wound management.