The two crystal structures of thrombin complexed with its most potent natural inhibitor hirudin and with the active-site inhibitor D-Phe-Pro-Arg-CH2Cl [Rydel, T.J. et al., J. Mol. Biol., 221 (1991) 583; Bode, W. et al., EMBO J., 8 (1989) 3467] were used as a basis to design a new inhibitor, combining the high specificity of the polypeptide hirudin with the simpler chemistry of an organic compound. In the new inhibitor, the C-terminal amino acid residues 53-65 of hirudin are linked by a spacer peptide of four glycines to the active-site inhibitor NAPAP (N alpha-(2-naphthyl-sulfonyl-glycyl)-DL-p-amidinophenylalanyl-piperi dine). Energy minimization techniques served as a tool to determine the preferred configuration at the amidinophenylalanine and the modified piperidine moiety of the inhibitor. The predictions are supported by the interaction energies determined for D- and L-NAPAP in complex with thrombin, which are in good agreement with experimentally determined dissociation constants. The conformational flexibility of the linker peptide in the new inhibitors was investigated with molecular dynamics techniques. A correlation between the Pl' position and the interactions of the linker peptide with the protein is suggested. Modifications of the linker peptide are proposed based on the distribution of its main-chain torsion angles in order to enhance its binding to thrombin.