In order to further understand the unusual specificity of thrombin activity, we have examined the interactions of thrombin with fibrinogen alpha-chain residues 27-50 by two complementary approaches. Using genetically engineered constructions, we have synthesized a fusion protein containing fibrinogen A alpha-residues 1-50 and seven variants of this protein with substitutions in alpha-residues 33-41. These fusion proteins were purified and analysed as thrombin inhibitors and substrates. Substitution of Phe35----Leu of Asp40----Gly substantially increased Ki, but all the fusion proteins were equally good substrates when assayed at concentrations equivalent to Ki. These results indicate that Phe35 and Asp40 are important for thrombin binding, but not catalysis. We have also synthesized a peptide analogous to fibrinogen alpha 27-50. This peptide was a competitive inhibitor of thrombin-catalysed cleavage of fibrinogen, but was not an inhibitor of hydrolysis of small chromogenic substrates. These data further indicate that residues alpha 27-50 are important in the thrombin-fibrinogen interaction.