A fractionation method applicable to different populations of cells in a suspension is reported. The separation was accomplished by subjecting the suspension to a resonant ultrasonic field and a laminar flow field propagating in orthogonal directions within a thin, rectangular chamber. Steady, laminar flow transports the cell suspension along the chamber, while the ultrasonic field causes the suspended cells to migrate to the mid-plane of the chamber at rates related to their size and physical properties. A thin flow splitter positioned near the outlet divides the effluent cell suspension into two product streams, thereby allowing cells that respond faster to the acoustic field to be separated from those cells that respond more slowly. Modeling of the trajectories of individual cells through the chamber shows that by altering the strength of the flow relative to that of the acoustic field, the desired fractionation can be controlled. Proof-of-concept experiments were performed using hybridoma cells and Lactobacillus rhamnosus cells. The two populations of cells could be effectively separated using this technique, resulting in hybridoma/Lactobacillus ratios in the left and right product streams, normalized to the feed ratio, of 6.9+/-1.8 and 0.39+/-0.01 (vol/vol), respectively. The acoustic method is fast, efficient, and could be operated continuously with a high degree of selectivity and yield and with low power consumption. (C) 2004 Wiley Periodicals, Inc.