Three potent competitive inhibitors of mammalian liver glyoxalase II, the S-(o-, m-, and p-nitrocarbobenzoxy)-glutathiones, have been synthesized and studied. The Ki values of the ortho, meta, and para isomers, as inhibitors of rat liver glyoxalase II, were 15, 9, and 6.5 microM, respectively. While showing marked competitive inhibition of glyoxalase II, the glutathione derivatives were almost inactive as inhibitors of glyoxalase I. For example, with the para isomer, [I]0.5 values for rat liver glyoxalase I and II were 925 and 12 microM, respectively. This is in marked contrast to other glyoxalase II competitive inhibitors, which in general are even more effective against glyoxalase I. The S-(o-, m-, and p-nitrocarbobenzoxy)glutathiones have found utility as affinity ligands for the purification of rat liver glyoxalase II and may well have use in the study of the glyoxalase enzymes in vivo.