Recombinant tick anticoagulant peptide (rTAP) is a highly selective inhibitor of blood coagulation factor Xa. rTAP has been characterized kinetically as a slow, tight-binding, competitive inhibitor of the enzyme. We used an approach consisting of both recombinant, site-directed mutagenesis and solid-phase chemical synthesis to generate 31 independent mutations in rTAP to identify those regions of the molecule which contribute to the specific, high-affinity binding interaction with factor Xa. Our results demonstrate that the four amino-terminal residues of rTAP constitute the primary recognition determinant necessary for the formation of the high-affinity enzyme-inhibitor complex. The Arg residue in position three is probably not interacting with the S1-specificity pocket of factor Xa in a substrate-like manner since substitution at this position with a D-Arg amino acid produced only a modest decrease in affinity (5-fold). An additional domain in the rTAP molecule located between residues 40 and 54 was identified as a probable secondary binding determinant. Interestingly, this region in rTAP shares significant amino acid sequence homology with a sequence in prothrombin immediately amino-terminal to the factor Xa cleavage site that generates meizothrombin. These observations indicate that specific segments within two different regions of the rTAP molecule contribute to the potent binding interaction between rTAP and factor Xa.