The role of the chondroitin sulfate moiety of thrombomodulin (TM) in the binding of thrombin to TM has been examined using fluorescent derivatives of thrombin. An anilinonaphthalene-6-sulfonic acid (ANS) dye was attached covalently to the active site histidine of thrombin via a D-Phe-Pro-Arg (FPR) linkage to form ANS-FPR-thrombin. When ANS-FPR-thrombin was titrated with TM lacking the chondroitin sulfate moiety (csf-TM), a monotonic and saturable increase in ANS emission intensity was observed that was consistent with the formation of a high affinity 1:1 thrombin-csf-TM complex. In contrast, titration of ANS-FPR-thrombin with intact TM containing the chondroitin sulfate resulted in a biphasic change in ANS-FPR-thrombin emission intensity that was consistent with each molecule of TM binding at least two molecules of ANS-FPR-thrombin with different affinities. This suggested that the second thrombin binds to TM via the chondroitin sulfate moiety. A direct interaction between thrombin and chondroitin sulfate was demonstrated by showing that chondroitin sulfate, cleaved and purified from TM, caused a saturable increase in ANS emission intensity upon addition to an ANS-FPR-thrombin sample. This spectral change was reversed by adding an excess of unmodified thrombin. The minimum Kd for the ANS-FPR-thrombin-chondroitin sulfate complex was approximately 20 nM, consistent with chondroitin sulfate being the lower affinity binding site on TM for thrombin. The titration of chondroitin sulfate into ANS-FPR-thrombin samples in the absence and presence of a TM fragment containing the fifth and sixth growth factor-like domains (GF5-6) showed that GF5-6 did not block chondroitin sulfate binding and that a GF5-6-thrombin-chondroitin sulfate ternary complex was formed. Thus, the chondroitin sulfate binds to thrombin somewhere other than anion-binding exosite I, and in doing so, alters the structure and/or environment of the active site more than 15A from the active site serine without detectably changing the conformation near Ser-195. Since excess TM and excess csf-TM increased the ANS emission intensity of ANS-FPR-thrombin to different extents (approximately 15 and approximately 80%, respectively), the chondroitin sulfate also influences the environment of the active site probe even when thrombin is bound to the higher affinity site on TM (GF5-6).