Glutathione (GSH) synthetase [L-gamma-glutamyl-L-cysteinyl:glycine ligase (ADP-forming), EC 6.3.2.3] catalyzes the final step in GSH biosynthesis. Mammalian glutathione synthetase is a homodimer with each subunit containing an active site. We report the detailed kinetic data for purified recombinant rat glutathione synthetase, It has the highest specific activity (11 mu mol/min/mg) reported for any mammalian glutathione synthetase, The apparent K-m values for ATP and glycine are 37 and 913 mu M, respectively. The Lineweaver-Burk double reciprocal plot for gamma-glutamyl substrate binding revealed a departure from linearity indicating cooperative binding. Quantitative analysis of the kinetic results for gamma-glutamyl substrate binding gives a Hill coefficient (h) of 0.576, which shows the negative cooperativity. Neither ATP, the other substrate involved in forming the enzyme-bound gamma-glutamyl phosphate intermediate, nor glycine, which at tacks this intermediate to form GSH, exhibit any cooperativity, The cooperative binding of gamma-glutamyl substrate is not affected by ATP concentration, Thus, mammalian glutathione synthetase is an allosteric enzyme.