Abstract Reactivity of sulfhydryl groups with 5 ,5'-dithiobis(2-nitrobenzoate) in 4 m urea and fluorescence emission of 1-anilinonaphthalene-8-sulfonate were used to probe changes in enzyme conformation. The substrate chorismate and the end product inhibitor tryptophan caused conformational alterations in anthranilate synthetase which were related to the mechanism of catalysis and inhibition, respectively. Tryptophan increased the rate of reaction of enzyme sulfhydryl groups while chorismate decreased the rate. Tryptophan and chorismate both decreased the fluorescence emission of enzyme-bound 1-anilinonaphthalene-8-sulfonate, but to different extents. The concentration dependence for changes in conformation correlated with saturation of catalytic and inhibitory sites with chorismate and tryptophan, respectively. Binding of chorismate and tryptophan was measured by equilibrium dialysis. Sites for 1 to 2 eq of each ligand were detected. Mg2+ reduced the dissociation constant for chorismate binding by 3- to 4-fold but did not change the number of available sites. Binding of tryptophan antagonized binding of chorismate and vice versa. Binding of tryptophan and chorismate appeared to be mutually exclusive although the data were not sufficiently precise to prove this point unequivocally. It is proposed that inhibition of anthranilate synthetase activity results when tryptophan binds to a regulatory site and maintains the enzyme in a conformation having poor affinity for substrates. The chorismate-dependent conformational change which was detected may facilitate binding of the second substrate glutamine.