Chondroitin-6-sulfate was oversulfated using chlorosulfonic acid-pyridine complex and isolated as the sodium salt. Infrared analysis of the native and oversulfated compound gave identical results in respect to the O{single bond}H stretching, hemiacetal stretching and S{double bond, long}O stretching. Absorption around 825 cm-1 was also present in both the compounds representing the sulfate group of galactosamine at equatorial C-6 position. The oversulfated compound showed a peak at 855 cm-1 representing the new sulfate group on axial C-4 position of galactosamine. 1H NMR studies confirmed the IR results and further showed that the oversulfated compound was a mixture of 61% of chondroitin-4,6-disulfate and 39% of the native compound. The oversulfated compound showed a 2.8-fold increase in sulfate content and a significant anticoagulant activity by doubling the prothrombin time of normal citrated human plasma using 9.5 μg of the sulfated compound while the native compound was inactive even at 2000 μg level. During in vitro studies using 0.05 M Tris buffer pH 7.35 containing physiological concentrations of NaCl (0.9%) the oversulfated compound gave a 1.8-fold enhancement of the activation of glutamic plasminogen (Glu-Plg) by tissue plasminogen activator (t-PA) and a 3.2-fold enhancement of Glu-Plg activation by urokinase (u-PA) in comparison to the control while the native compound or the unfractionated heparin were less active. © 2007.