A high-affinity thrombin-binding site in an alternately processed fibrinogen variant, the γA/γ' isoform, is characterized in this report. The binding site has been shown to be situated between γ' 414 and 427, and Tyr418 and 422 in this part of the γ' chain are known to be sulfated. A synthetic peptide corresponding to the γ' chain carboxyl terminus is shown to bind thrombin with a K d=0.63±0.16 μmol L -1. Maximum binding of this peptide requires negative charges on Tyr418 and 422. Competitive binding studies with hirudin peptides, heparin and DNA aptamers specific for thrombin exosites I or II indicate thrombin binds to the γ' peptide via exosite II. Thus, thrombin binding to the γ' chain leaves exosite I and the active site accessible to substrates. This may explain why fibrin-bound thrombin can retain enzymatic activity, and why fibrin-bound thrombin is heparin-resistant.