A recombinant variant of hirudin, the blood‐clotting inhibitor of the leech Hirudo medicinalis, has been characterized employing spectroscopic and hydrodynamic techniques. Conditions have been defined for efficient reconstitution of the native, disulfide‐bonded inhibitor from completely unfolded, reduced polypeptide chains.The spectral properties of the native inhibitor are consistent with previous results on the solution structure of hirudin. Extremely low circular dichroism in the far ultraviolet () indicates a very low content of regular secondary structure. Although both tyrosine residues of the recombinant inhibitor titrate around pH 10.6, typical for solvent‐exposed tyrosines, fluorescence emission and near‐ultraviolet circular dichroism suggest that at least one of the tyrosines is partially shielded from solvent quench, and immobilized in an asymmetric environment. Reversible thermal unfolding of hirudin around 65° C is indicated by the disappearance of its dichroic absorption in the near ultraviolet and by a fourfold increase in ellipticity at 225 nm. The transition can be approximated by a two‐state model with a transition enthalpy of Hvan't Hoff= 159 kJ/mol and a transi
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