A series of bivalent thrombin inhibitors was synthesized, consisting of a dphenylalanylprolylN?(methyl) arginyl active site blocking segment, a fibrinogen recognition exosite inhibitor part, and a peptidic linker connecting these fragments. The methylation of the P1 amino acid led to a moderate decrease in affinity compared with the unmethylated analog. In addition, it prevented the thrombin catalyzed proteolysis, independent of the P1' amino acid used. This is a significant advantage compared to the original hirulogs, which strictly require a proline as P1' amino acid to reduce the cleavage Cterminal to the arginyl residue. Several analogs were prepared by incorporation of different P1' amino acids found in natural thrombin substrates. The most potent inhibitor was I-11 [dCha ProN(Me)ArgThr(Gly)[5]DYEPIPEEAChadGlu] with a K of 37 pM. I-11 is highly selective and no inhibition of the related serine proteases trypsin, factor Xa and plasmin was observed. The stability of I-11 in human plasma in vitro was strongly improved compared to hirulog-1. In addition, a significantly reduced plasma clearance of I-11 was observed after intravenous injection in rats. Results from molecular modeling suggest that a strong reorganization of the hydrogen bonds in the active site of thrombin may result in the proteolytic stability found in this inhibitor series.