Thrombin (IIa)-cleavage of fibrinogen (FBG) to form polymerized fibrin promotes endothelial cell spreading, proliferation, and von Willebrand factor release, requiring the exposure of the beta 15–42 domain. Studies reported here indicate that IIa-cleavage of fibrinopeptide B enhances exposure of a heparin binding domain at the beta 15–42 neo-N- terminus of fibrin. Crossed immunoelectrophoresis showed heparin- induced mobility shifts indicative of complexing with FBG and with N- terminal CNBr fragments of FBG (NDSK) and of fibrin (IIa-NDSK), but not evidence of heparin complexing with FBG lacking B beta 1–42 or with FBG fragments D and E was seen. Elution from heparin-agarose with a linear gradient of NaCl showed that bound portions of both intact FBG and D fragments eluted below physiologic salt concentrations, whereas E3 fragments lacking B beta 1–53 did not bind. NDSK bound with higher affinity than did intact FBG, whereas binding of IIa-NDSK was maximal in this system. Binding of fibrin(ogen) to heparin agarose was saturable as well as inhibitable in a dose-dependent manner with both FBG and heparin. Scatchard analysis indicated a single class of binding site, with dissociation constants (kd) of 0.3 mumol/L for IIa-NDSK, 0.8 mumol/L for NDSK, and 18 mumol/L for FBG. Immobilized fibrin had twofold more heparin binding sites than did immobilized FBG and required a 5.5-fold higher concentration of heparin to inhibit by 50% the binding of labeled heparin. Together, the results indicate that IIa- cleavage results in enhanced exposure of two heparin binding domains (HBDs) with approximately threefold higher affinity in fibrin than in FBG. Synthetic peptide beta 15–42 showed highest binding to heparin- agarose followed by B beta 1–42, whereas peptides beta 18–31, beta 18– 27, and beta 24–42 did not bind. Thus, the primary structure of beta 15– 42 is required for specificity of heparin binding. Basic residues within the beta 15–32 region segregate primarily to one side of an alpha-helix in a helical wheel diagram, as is typical for authentic HBDs. Desulfated heparin and heparan sulfate bound more fibrin(ogen) than did other proteoglycans; however, heparin bound sixfold more Ila- NDSK than NDSK. These results confirm that fibrin binds to heparin with higher affinity than does FBG and that fibrin binding is not solely dependent on charge interactions of beta 15–42 with the negatively charged glycosaminoglycan.