The reaction of N-ethylmaleimide with chorismate mutase/prephenate dehydratase (chorismate pyruvatemutase/prephenate hydrolyase (decarboxylating) EC 5.4.99.5/EC 4.2.1.51) from Escherichia coli K12, which leads to the preferential inactivation of the prephenate dehydratase activity (Gething, M-J.H. and Davidson, B.E. (1977) Eur. J. Biochem. 78, 111-117), was found to involve only the sulphydryl groups of the enzyme. Determination of the reactivities of the four different cysteine residues indicated that the reaction was not specific for a single residue, although two residues (Cys-216 and Cys-374) were more reactive than the others. The amount of inhibition of the prephenate dehydratase activity approximated in extent to the sum of the stoichiometries of the individual reactions of N-ethylmaleimide with these two cysteine residues. In the presence of either phenylpyruvate, the product of the prephenate dehydratase activity, or cis-aconitate, a competitive inhibitor with respect to prephenate, the prephenate dehydratase activity was substantially protected from inactivation. This protection was concomitant with a significant decline in the reactivities of both Cys-216 and Cys-374. These results are interpreted as indicating that both of these cysteine residues are at, or near to, the prephenate dehydratase active site and are possibly essential for the prephenate dehydratase activity of the enzyme.