Aconitases have recently regained much attention, because one member of this family, iron regulatory protein-1 (IRP-1), has been found to play a dual role as a cytoplasmic aconitase and a regulatory RNA-binding protein. This finding has highlighted a novel role for Fe-S clusters as post-translational regulatory switches. We have aligned 28 members of the Fe-S isomerase family, identified highly conserved amino acid residues, and integrated this information with data on the crystallographic structure of mammalian mitochondrial aconitase. We propose structural and/or functional roles for the previously unrecognized conserved residues. Our findings illustrate the value of detailed protein sequence analysis when high-resolution crystallographic data are already available.