Nonsubstrate interactions of thrombin with fibrin play an important role in modulating its procoagulant activity. To establish the structural basis for these interactions, we crystallized d-Phe-Pro-Arg-chloromethyl ketone-inhibited human thrombin in complex with a fragment, E(ht), corresponding to the central region of human fibrin, and solved its structure at 3.65-A resolution. The structure revealed that the complex consists of two thrombin molecules bound to opposite sides of the central part of E(ht) in a way that seems to provide proper orientation of their catalytic triads for cleavage of fibrinogen fibrinopeptides. As expected, binding occurs through thrombin's anion-binding exosite I. However, only part of it is involved in forming an interface with the complementary negatively charged surface of E(ht). Among residues constituting the interface, Phe-34, Ser-36A, Leu-65, Tyr-76, Arg-77A, Ile-82, and Lys-110 of thrombin and the A alpha chain Trp-33, Phe-35, Asp-38, Glu-39, the B beta chain Ala-68 and Asp-69, and the gamma chain Asp-27 and Ser-30 of E(ht) form a net of polar contacts surrounding a well defined hydrophobic interior. Thus, despite the highly charged nature of the interacting surfaces, hydrophobic contacts make a substantial contribution to the interaction.