Microsomal glutathione transferase is an abundant liver protein that can be activated by thiol reagents. It is not known whether the activation is associated with changed binding properties of the enzyme. Therefore the binding of GSH and an inhibitor to rat liver microsomal glutathione transferase was studied by use of equilibrium dialysis and equilibrium partition in a two-phase system. The radioactive substrate glutathione and an inhibitor (glutathione sulphonate) give hyperbolic binding isotherms with a stoichiometry of 1 mol per mol of enzyme (i.e. 1 molecule per homotrimer). Glutathione had an equilibrium binding constant of 18 microM. Competition experiments involving glutathione sulphonate showed that it could effectively displace GSH. These and kinetic studies showed that the Kd and Ki for glutathione sulphonic acid are close to 10 microM. No change in these parameters was obtained after N-ethylmaleimide activation of the enzyme. Thus activation does not result from changes in binding affinity to GSH.