A separation procedure was developed using S-Sepharose cation exchange chromatography under mildly acidic conditions to establish the effect of 70% (v/v) ethanol extractable proteins on bread-making quality. The separation of a 70% (v/v) ethanol extract of gluten was scaled up successfully from 3 mg to 60 g protein. The use of denaturing and dissociating agents, such as urea or guanidine-HCl, was unnecessary. The pilot-scale fractionation yielded five fractions that were bound to, and eluted from, the column. The fractions differed in gliadin composition as evidenced by lactate-PAGE, SDS-PAGE and RP-HPLC. The fractions were virtually free of lipid. Isolated fractions were evaluated for their effects on bread-making quality using a pan loaf baking test. The unbound fraction (D) contained lipids, mainly mono- and digalactosyldiacylglycerols, and it increased (cv. Obelisk flour) or decreased (cv. Camp Rémy flour) loaf volume at additions of up to 0·5% on flour weight. At higher levels of addition it had a strong negative effect on loaf volume. The unfractionated extract (containing some fraction D) or the recombined fractions improved loaf volume. All individual fractions improved loaf volume, but to different extents. For four out of five fractions the improvement corresponded to the statistical prediction of loaf volume by RP-HPLC gliadin peak areas.