Escherichia coil glyoxalase I (GU) is the first example of an isomerase that is maximally activated by Ni2+. In contrast with GlxI enzymes from other sources that are Zn enzymes, E. coli GlxI is not active with Zn bound. Structural details from X-ray absorption spectroscopic analyses are reported for the active site Ni center and the Zn-substituted enzyme. The available data regarding the structure of the active Ni site are consistent with a six-coordinate Ni(Glu)(2)(His)(2)(OH2)(2) site in the enzyme.