Zein is a group of alcohol-soluble corn proteins, which consists of several individual proteins. A single-step gel filtration chromatography method was developed to fractionate individual zeins from ethanol extracts of whole corn. A Superdex prep 75 column was used with different mobile phases to fractionate the zeins, which were analyzed by SDS-PAGE and UV spectrophotometry. With 70% aqueous ethanol as the mobile phase, fractions containing a mixture of alpha-zein/beta-zein and alpha-zein/delta-zein were obtained. With ammonium bicarbonate added to the 70% ethanol mobile phase, it was possible to obtain beta-zein and delta-zein fractions devoid of other proteins. However, all fractions containing alpha-zein also contained minor amounts of delta-zein and/or beta-zein. Almost all fractions also contained non-protein impurities.