Loading DNA onto microbubbles is desirable for applications in gene therapy involving localized delivery with ultrasound. In such applications, cationic lipids are mixed into the monolayer shell to electrostatically bind to negatively charged genetic material. The present work focuses on experimental results detailing the adsorption of DNA molecules onto the microbubble shell. The microbubbles were dispersed in aqueous media and consisted of perfluorobutane gas encapsulated in a monolayer shell of distearoyl-trimethylammoniumpropane (DSTAP), disteroyl-phosphatidylcholine (DSPC) and poly(ethylene glycol)-40 stearate (PEG40S). First, we looked at charge effects on microbubble dispersion. Microbubble stability was robust up to 40 mol% DSTAP and depended strongly on ionic strength. Fortuitously, production yield and stability were optimal at physiological ionic strength (10 mM NaCl). Next, we investigated the adsorption of linear and plasmid DNA onto the cationic shell. DNA loading was heterogeneous and occurred primarily on the condensed phase domains, suggesting that DSTAP is miscible with DSPC, but not PEG40S, and opening the possibility to engineer distinct domains for gene loading and receptor targeting. The amount of adsorbed DNA increased linearly with mole fraction of DSTAP. Finally, we employed layer-by-layer assembly with poly-L-lysine (PLL) to enhance DNA loading. For the first paired layer, the amount of adsorbed PLL correlated directly with the amount of adsorbed DNA. Multilayer buildup was demonstrated by zeta potential measurements, fluorescence microscopy, UV spectroscopy and flow cytometry. The multilayers exhibited two growth stages and were heterogeneously distributed over the microbubble surface. For five paired layers, DNA loading capacity was enhanced by over tenfold. These results illustrate how microbubbles can be used for high-throughput studies of the interactions of biomolecules with Langmuir monolayers and, furthermore, they suggest that lipid mixing and multilayer formation on the surface can be engineered to form superior contrast agents for ultrasonic gene therapy. We acknowledge the support by NIH (R01 CA 103828).