The nature of the binding determinants used in the interaction of glutathione-based derivatives and bovine liver glyoxalase II (S-(2-hydroxyacyl)glutathione hydrolase, EC 3.1.2.6) has been investigated. Linear competitive inhibition was observed for S-blocked and S,N-blocked glutathiones with bovine liver glyoxalase II (molecular weight 22 500 by sodium dodecyl sulphate polyacrylamide gel electrophoresis; pI = 7.48 by analytical isoelectric focussing). There is a significant hydrophobic region on the enzyme to bind substituents around the sulphydryl-derived moiety of the substrate--a hydrophobic S-site. However, there is no evidence for binding of the N-site of the substrate (or inhibitor) to glyoxalase II. In contrast to glyoxalase I, there is no linkage between binding forces used at the S- and N-sites. Binding of S,N-dicarbobenzoxyglutathione is pH-dependent, showing dependence on an ionisation with pKapp approximately equal to 7.2 (binding more tightly at higher pH), as is the kcat value (pKapp approximately equal to 7.8) for S-D-lactoylglutathione.