Ultrasound is commonly used in the preparation of unilamellar liposome dispersions and is often considered for cell membrane disruption for drug delivery or DNA transfection applications. To better understand the physical and chemical properties of lipid membranes that render them susceptible to ultrasonic permeabilization, the roles of temperature, lipid composition (cholesterol and PEG-lipid content), and liposome size have been studied. The results of these studies suggest that lipid packing is very important to ultrasound responsiveness; surprisingly, cohesive energy and tensile strength are not. Taken together, the experimental results implicate a defect-mediated permeabilization mechanism, rather than pore formation or membrane tearing. The implications of this work for drug release from liposomes and ultrasound mediated DNA transfection are discussed.