Abstract Both glyoxalase enzymes from various sources are inhibited by nucleotides, nucleosides and a series of structurally related compounds in an apparently cooperative manner. In general, aromatic compounds, which contain heterocyclic nitrogen and/or have amino groups on the aromatic ring, are effective inhibitors of these enzymes. Since intracellular concentrations of some of the nucleotides are significantly higher than the concentrations needed to inhibit both enzymes completely, the inhibitions observed may have physiological significance.