The thiol-oxidizing agent "diamide" (CH3)2NCON equal to NCON(CH3)2 was used to isolate mutants of Escherichia coli K 12 deficient in the biosynthesis of glutathione. A colony-colour technique has been developed for identification of colonies of these mutants. Four glutathione-deficient mutants were isolated. They show normal growth rates in minimal medium without GSH supplementation, indicating that glutathione is not involved in essential metabolic process. In one mutant, glutathione synthetase was entirely inactive. Three mutants were deficient in gamma-glutamylcysteine synthetase; in two of them, this resulted in a complete lack of GSH. These mutants were found to be more susceptible than their parent strains to a wide rang of chemical agents, but did not show a greater sensitivity to X-rays. It must be concluded that the protective role of glutathione is only significant when a chemical challenge is present.