Metal dissociation constants for glyoxalase I from human erythrocytes were determined by use of nitrilotriacetic acid as a metal buffer. The constants for Zn2+, Co2+, Mn2+, and Mg2+ were (2.7 +/- 0.3) X 10(-11) M, (3.0 +/- 0.8) X 10(-10) M, (4.9 +/- 0.5) X 10(-9) M, and (1.0 +/- 0.2) X 10(-6) M, respectively, demonstrating that the natural cofactor, Zn2+, has the highest affinity for the apoprotein. The results are consistent with the proposal of nitrogen and oxygen atoms as ligands to the metal in the active site of glyoxalase I. In the application of the metal buffer technique, it was found that both 1:1 and 1:2 complexes of the metal ions and nitrilotriacetic acid have to be considered.