Microbubbles used as contrast agents for ultrasound imaging, vectors for targeted drug delivery and vehicles for metabolic gas transport require better size control for improved performance. Mechanical agitation is the only method currently available to produce microbubbles in sufficient yields for biomedical applications, but the emulsions tend to be polydisperse. Herein, we describe a study to generate lipid-coated, perfluorobutane-filled microbubbles and isolate their size fractions based on migration in a centrifugal field. Polydispersity of the freshly sonicated suspension was characterized by particle sizing and counting through light obscuration/scattering and electrical impedance sensing, fluorescence and bright-field microscopy and flow cytometry. We found that the size distribution was multimodal. Smaller microbubbles were more abundant. Differential centrifugation was used to successfully isolate the 1-2 and 4-5 mum diameter fractions. Isolated microbubbles were stable over two days. After two weeks, however, more dilute suspensions (<1 vol%) were susceptible to Ostwald ripening. For example, 4-5 mum microbubbles disintegrated into 1-2 mum microbubbles. This latter observation indicated the existence of an optimally stable diameter in the 1-2 mum range for these lipid-coated microbubbles. Overall, differential centrifugation provided a rapid and robust means for size selection and reduced polydispersity of lipid-coated microbubbles.