1. Six kinds of unsaturated disaccharides were prepared by enzymatic digestion of heparin and heparan sulfate with heparitinases I0 and IV, and subsequent column chromatography. They were identified by HPLC showing good separation from each other. 2. The content of each unsaturated disaccharide fraction was determined colorimetrically, and found to range from 130.7 to 722.3 mumol. 3. Molecular extinction coefficient of each unsaturated disaccharide was calculated from absorbance at a wavelength of around 230 nm where a peak appeared on the ultraviolet spectrum of each disaccharide solution at pH 2. The values varied from 6000 to 6600.