Rice seed storage glutelin could be separated into three acidic (alpha-) and three basic (beta-) subunit components by SDS-PAGE using 15%-25% gradient acrylamide gel. More than 13 acidic and 14 basic bands may be distinguished with an improved iso-electric focus electrophoresis system by adjusting Ampholine ratios. Combine these two sensitive electrophoresis systems resulting in two-dimensional electrophoresis giving high resolution of glutelin leading the yield of single polypeptide. The improved electrophoresis system is helpful in identifying rice glutelin variation and for further studies of glutelin biochemistry.