Uniform-sized agarose beads with diameters less than 10 mu m and agarose content as high as 14wt% were prepared by premix membrane emulsification. Agarose aqueous solution was used as the water phase. A mixture of liquid paraffin and petroleum ether containing hexaglycerin penta ester (PO-500) was used as the oil phase. The water phase was mixed with the oil phase at 60 degrees C and a coarse W/O emulsion was produced in a homogenizer. Then, the coarse emulsion was extruded through a hydrophobic membrane under high pressure to form an emulsion, which was slowly cooled under gentle agitation to form gel beads. The effects of preparation conditions on emulsification results were investigated and it showed that the pressure, number of passes, petroleum ether/liquid paraffin (v/v) in the oil phase, the concentration of PO-500 and concentration of agarose in the water phase, all affected the size and uniformity; coarse emulsion did not affect the emulsification results. The coefficient variation (C.V.) of agarose beads under optimal preparation conditions was 9.8%. This method realized microbeads with both uniform sizes and high agarose contents that are difficult to be prepared by conventional emulsion methods. (c) 2008 Elsevier B.V. All rights reserved.