The purpose of this study was to further understand the mechanisms involved in ultrasound-mediated delivery of DNA (sonoporation); in particular, to understand how a plasmid should be formulated with an ultrasound contrast agent (UCA). Different polymer adjuvant-UCA combinations were formulated, and their impact on in vitro DNA transfection, was determined, under various experimental conditions. When present in the medium surrounding a cell suspension, and in the presence of a plasmid encoding for the green fluorescent protein (GFP), expression following sonoporation was increased by more than 1.5-fold compared to that achieved in control experiments (without the adjuvants). The effects of the adjuvants were not influenced by the nature of the UCA, nor by that of the transfected cells; in contrast, the adjuvant concentrations, their physico-chemical properties, and the manner in which they were used, did have an impact on transfection. Close association of the adjuvants to the UCA inhibited their action, suggesting that these substances must have access to the cell membrane to be effective. Indeed, Pluronic F127 appeared to improve the efficacy of transfection (percentage of GFP-positive cells and cell viability), via fluidization of the cell membrane, perhaps facilitating thereby the formation of transient pores and their re-sealing. The mechanism of action of polyethylene glycols, on the other hand, remains unclear.