AbstractGlutaredoxins (Grxs) are glutathione‐dependent oxidoreductases that belong to the thioredoxin superfamily catalyzing thiol‐disulfide exchange reactions via active site cysteine residues. Focusing on the human dithiol glutaredoxins having a C‐X‐Y‐C active site sequence motif, the redox potentials of hGrx1 and hGrx2 were determined to be −232 and −221 mV, respectively, using a combination of redox buffers, protein–protein equilibrium and thermodynamic linkage. In addition, a nonactive site disulfide was identified between Cys28 and Cys113 in hGrx2 using redox buffers and chemical digestion. This disulfide confers nearly five kcal mol−1additional stability by linking the C‐terminal helix to the bulk of the protein. The redox potential of this nonactive site disulfide was determined to be −317 mV and is thus expected to be present in all but the most reducing conditionsin vivo. As all human glutaredoxins contain additional nonactive site cysteine residues, a full phylogenetic analysis was performed to help elucidate their structural and functional roles. Three distinct groups were found: Grx1, Grx2, and Grx5, the latter representing a highly conserved group of monothiol glutaredoxins having a C‐G‐F‐S active site sequence, with clear homologs from bacteria to human. Grx1 and Grx2 diverged from a common ancestor before the origin of vertebrates, possibly even earlier in animal evolution. The highly stabilizing nonactive site disulfide observed in hGrx2 is found to be a conserved feature within the deuterostomes and appears to be the only additional conserved intramolecular disulfide within the glutaredoxins. Proteins 2007. © 2007 Wiley‐Liss, Inc.