γ-Glutamyl transpeptidase has been partially purified from kidneys of adult male rats by solubilization with deoxycholate and chromatography in the presence of Triton X-100. A new, sensitive assay has been developed to study the reaction of glutathione with methionine in the micromolar range. This assay was found to differ from those using γ-glutamylnaphthylamide as substrate in the activation of the enzyme by anions and cations, and in terms of the nature of the reaction catalyzed by the enzyme. The kinetics of the transpeptidation reaction have been studied with the substrates glutathione and methionine. The pattern of inhibition caused by the product cysteinylglycine suggested that the reaction was non-sequential. The Michaelis constants for glutathione and methionine were of the order of 40 μM and 3 mM, respectively.