Glyoxalase I from human erythrocytes was studied by use of the strong reversible competitive inhibitor S - p -bromobenzylglutathione. Replacements of cobalt, manganese and magnesium for the essential zinc in the enzyme were made by a new procedure involving 10% methanol as a stabilizer of the enzyme. The K m value for the adduct of methylglyoxal and glutathione was essentially unchanged by the metal substitutions, whereas the inhibition constant for S - p -bromobenzylglutathione increased from 0.08μ m for the Zn-containing enzyme to 1.3, 1.7 and 2.4μ m for Co-, Mn- and Mg-glyoxalase I respectively. Binding of the inhibitor to the enzyme caused quenching of the tryptophan fluorescence of the protein, from which the binding parameters could be determined by the use of non-linear regression analysis. The highest dissociation constant was obtained for apoenzyme (6.9μ m ). The identity of the corresponding kinetic and binding parameters of the native enzyme and the Zn 2+ -re-activated apoenzyme and the clear differences from the parameters of the other metal-substituted enzyme forms give strong support to the previous identification of zinc as the natural metal cofactor of glyoxalase I. Binding to apoenzyme was also shown by the use of S - p -bromobenzylglutathione as a ligand in affinity chromatography and as a protector in chemical modification experiments. The tryptophan-modifying reagent 2-hydroxy-5-nitrobenzyl bromide caused up to 85% inactivation of the enzyme. After blocking of the thiol groups (about 8 per enzyme molecule) 6.1 2-hydroxy-5-nitrobenzyl groups were incorporated. Inclusion of S - p -bromobenzylglutathione with the modifying reagent preserved the catalytic activity of the enzyme completely and decreased the number of modified residues to 4.4 per enzyme molecule. The findings indicate the presence of one tryptophan residue in the active centre of each of the two subunits of the enzyme. Thiol groups appear not to be essential for catalytic activity. The presence of at least two categories of tryptophan residues in the protein was also shown by quenching of the fluorescence by KI.