A discontinuous Tris-Cl/acetate (OAc) buffer system, unprecedently containing OAc as the trailing constituent, and operative in polyacrylamide gel electrophoresis (PAGE) at low polyacrylamide concentration (T = 4.8%) is described in the paper. The characteristics of the electrophoretic system are illustrated by the resolution of fluorescent 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS)-labeled malto-oligosaccharides and dextran homopolymers. In this buffer system, the resolving phase is constituted by Tris-OAc behind a moving boundary formed between the leading chloride ion of Tris-HCl gel buffer and the trailing OAc ion provided by a catholyte of NH4OAc. In contrast with the results obtained with Tris-Cl/glycinate buffer commonly used in electrophoresis, or with Tris-Cl/borate, the best resolution of the glucose oligomers containing 1-4 glucose units in Tris-OAc, pH 8.8, ionic strength of 0.08, was obtained at 4.8% polyacrylamide concentration, using 0.5 m NH4OAc, pH 9.5 as the catholyte. Under those conditions, the ANTS-glucose oligomers were separated with mobilities decreasing from glucose to maltohexaose. The linear Ferguson plots (log relative mobility, R-f, vs.%T) of the glucose oligomers show that the surface net charge of those oligomers is inversely related to their sizes, given by the slopes, K-R, of the plots. The molecular weight of the oligomers is directly but nonlinearly related to K-R. The novel electrophoretic system illustrated here for separation of short ANTS-saccharides can be potentially applied to the resolution of other biomolecules such as rapidly migrating DNA, peptides or proteins.