Grx5 defines a family of yeast monothiol glutaredox- ins that also includes Grx3 and Grx4. All three proteins display significant sequence homology with proteins found from bacteria to humans. Grx5 is involved in iron/ sulfur cluster assembly at the mitochondria, but the function of Grx3 and Grx4 is unknown. Three-dimen- sional modeling based on known dithiol glutaredoxin structures predicted a thioredoxin fold structure for Grx5. Positionally conserved amino acids in this glu- taredoxin family were replaced in Grx5, and the effect on the biological function of the protein has been tested. For all changes studied, there was a correlation between the effects on several different phenotypes: sensitivity to oxidants, constitutive protein oxidation, ability for respiratory growth, auxotrophy for a number of amino acids, and iron accumulation. Cys60 and Gly61 are essen- tial for Grx5 function, whereas other single or double substitutions in the same region had no phenotypic ef- fects. Gly115 and Gly116 could be important for the for- mation of a glutathione cleft on the Grx5 surface, in contrast to adjacent Cys117. Substitution of Phe50 alters the ␤-sheet in the thioredoxin fold structure and inhib- its Grx5 function. None of the substitutions tested affect the structure at a significant enough level to reduce protein stability.