C-Terminal fragment analogues of the leech protein hirudin or the related protein hirudin PA block thrombin's cleavage of fibrinogen. Three series of synthetic peptides were synthesized to study the effects of sulfation in hirudin-derived peptides. Potency of hirudin analogues increased with p-(amino)Phe63, p-(aminosulfonate)Phe63, and p-(sulfate)Tyr63 substitution in place of Tyr63. Sulfation of Tyr56, which in hirudin is normally Phe, resulted in a loss of 1 order of magnitude in potency. The sulfation of Tyr64 of the hirudin PA related analogue resulted in increased potency as for the hirudin analogue. However, in this series the p-(amino)Phe64 and p-(amino-sulfonate)Phe64 did not have increased potency. In addition to these positional effects, replacing all the Glu residues with (O-sulfato)Ser yielded an analogue with full antithrombin potency.