To describe the role of the lysyl binding site in the interaction of tissue-type plasminogen activator (t-PA, FGK1K2P) with a forming fibrin clot, we performed binding experiments with domain deletion mutants GK1K2P, K2P, and the corresponding point mutants lacking the lysyl binding site in the absence and the presence of ε-amino caproic acid (EACA). Occupation of the lysyl binding site in the K2 domain with EACA has a pronounced effect on the binding of FGK1K2P to a fibrin clot (C50 = 77 ± 11 nM versus 376 ± 45 nM with EACA). Deleting the lysyl binding site in the K2 domain (substitution D236N) also impairs fibrin binding but to a lesser extent (C50 = 169 ± 20 nM). Although the binding of K2P to a fibrin clot is weak (C50 = 1163 ± 490 nM), it still is 2 orders of magnitude stronger than the binding of EACA to K2P. Therefore it was surprising to find that deletion of the lysyl binding site in K2P completely abolishes fibrin binding. Even when both the F domain and the lysyl binding site were deleted, considerable fibrin binding is still observed (C50 = 557 ± 126 nM), suggesting other than F and K2- mediated interactions. The binding of FGK1K2P, FGK1K2P (D236N), GK1K2P, and GK1K2P (D236N) to fibrin could be competitively inhibited by FGK1K2P and K2P, indicating that all molecules recognize the same interaction sites on a fibrin clot. Based on these results, a new model for the interaction of t-PA with a forming fibrin clot is proposed. The fibrin binding sites in t-PA are not confined to the F and K2 domain. The main role of the lysyl binding site in the K2 domain of t-PA appears indirect rather than direct, most likely stabilizing a conformation favorable for fibrin binding.