Covalent incorporation (cross-linking) of plasmin inhibitor alpha(2)-antiplasmin (alpha(2)-AP) into fibrin clots increases their resistance to fibrinolysis. We hypothesized that alpha(2)-AP may also interact noncovalently with fibrin prior to its covalent cross-linking. To test this hypothesis, we studied binding of alpha(2)-AP to fibrin(ogen) and its fragments by an enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance. The experiments revealed that alpha(2)-AP binds to polymeric fibrin and surface-adsorbed fibrin(ogen), while no binding was observed with fibrinogen in solution. To localize the alpha(2)-AP-binding sites, we studied the interaction of alpha(2)-AP with the fibrin(ogen)-derived D(1), D-D, and E(3) fragments, and the recombinant alphaC region and its constituents, alphaC connector and alphaC domain and its subdomains, which together encompass practically the whole fibrin(ogen) molecule. In the ELISA, alpha(2)-AP bound to immobilized D(1), D-D, alphaC region, alphaC domain, and its C-terminal subdomain. The binding was Lys-independent and was not inhibited by plasminogen or tPA. Furthermore, the affinity of alpha(2)-AP for D-D was significantly increased in the presence of plasminogen, while that to the alphaC domain remained unaffected. Altogether, these results indicate that the fibrin(ogen) D region and the C-terminal subdomain of the alphaC domain contain high-affinity alpha(2)-AP-binding sites that are cryptic in fibrinogen and exposed in fibrin or adsorbed fibrinogen, and the presence of plasminogen facilitates interaction of alpha(2)-AP with the D regions. The discovered noncovalent interaction of alpha(2)-AP with fibrin may contribute to regulation of the initial stage of fibrinolysis and provide proper orientation of the cross-linking sites to facilitate covalent cross-linking of alpha(2)-AP to the fibrin clot.