Heparin-derived oligosaccharides, prepared by using flavobacterial heparinase, having a high degree of heterogeneity (sequence variability) were resolved into sharp well-defined bands by using polyacrylamide gel electrophoresis (PAGE). The use of a stacking gel and a high-density-pore-gradient resolving gel was primarily responsible for the success of this separation. Low-Mr standards of known structure and having a degree of polymerization (dp) 2-6 were used to establish that the separation on gradient PAGE was primarily dependent on molecular size. High-Mr oligosaccharides (dp 8-20) were prepared using strong-anion-exchange h.p.l.c. and were used to help characterize the gradient PAGE separation. Kinetic profiles were obtained for the depolymerization of heparin and heparan sulphate with heparinase and heparitinase respectively. The utility of this approach in sequencing oligosaccharides derived from glycosaminoglycans is discussed.