ω-Heparin isolated from whale organs was digested with an eliminase [heparin lyase, EC 4.2.2.7] prepared from crude heparinase, which was obtained from α-heparin-adapted flavobacteria. The digestion products were then fractionated into 16 fractions by ion-exchange column chromatography on Dowex-1. Subsequently, the resulting fractions were purified by rechromatography on Dowex-1 and or by preparative paper chromatography, yielding almost quantitatively purified compounds in fractions 2, 4, 5, 6, and 12. In addition, compounds 10a and 15a were isolated in a homogeneous state from fractions 10 and 15. Compounds 2, 4, 5, 6, 10a, 12, and 15a were characterized and identified as N-acetylglucosamine,Δ4, 5-hexosyluronic acid-N-acetylglucosamine, N-sulfated glucosamine, Δ4,5-hexosyluronic acid-N-sulfated glucosamine, N-, 6-O-disulfated glucosamine, and most probably sulfated Δ4, 5-hexosyluronic acid-N-sulfated glucosamine and sulfated Δ4,5-hexosyluronic acid-N-, 6-O-disulfated glucosamine, respectively, by chemical and enzymic studies together with infrared and ultraviolet spectral analyses. Furthermore, the major fractions (fractions 12, 13, 14, and 15) were each shown to contain two or three sulfate residues per unsaturated disaccharide unit. The total yield of unsaturated oligosaccharides was 66.6% of the starting material, while the major fractions accounted for 48.2%. In agreement with previous data, the major glucosaminidic linkages of ω-heparin appear to be 1-4. The present study suggests that ω-heparin has a more complex structure than α-heparin.