The insulin degrading enzyme (IDE), a nonlysosomal enzyme involved in the metabolism of internalized insulin, is a member of a new family of metalloproteases which has an HXXEH active site motif. We have previously shown that both His108 and Glu111 within the HXCEH domain of human IDE are necessary for catalytic activity. Comparison to the prototypic zinc metalloprotease thermolysin, which contains an inversion of this motif, would predict that His112, as well as a downstream glutamate, serves as the second and third zinc ligands of IDE. To examine the role of His112, we mutated this residue to glutamine, leucine, or arginine. To identify a downstream zinc ligand, we substituted a glutamine for glutamate at either Glu182 or Glu189, both of which are conserved in human, rat, and Drosophila IDE. Vectors containing wild type or mutant IDE genes were transfected into COS cells, and the enzymes were analyzed for insulin degradation, insulin cross-linking, and zinc binding. Our results suggest that His108, His112, and Glu189 are the zinc ligands of human IDE, and Glu182 can influence zinc binding. In addition to a catalytic role, zinc binding to these residues appears to play a role in stabilizing the structure of the enzyme.