The concentration of glutathione (GSH) in bacteria is many-fold less than in mammalian cells except for Escherichia coli, where the GSH level is similar to that of mammalian tissues. On the basis of our observation that GSH in a B strain, ATCC 29682, was reduced (> 80%) by exposure to oxidants while a K-12 strain (AB 1157) was minimally affected (< 20%), we constructed a B strain GSH-deficient mutant that exhibited antioxidant enzyme activities similar to the wild strain. We successfully transduced the gsh A::Tn10Km allele from JTG-10, a GSH-deficient K-12 strain, to ATCC 29682, the GSH-sufficient B strain. Compared with ATCC 29682, the growth of the GSH-deficient B mutant, designated RCI-1, was more sensitive to the presence of thiol-reactive chemicals. However, no difference was found between GSH-sufficient and -deficient strains in lethality following exposure to the same thiol-reactive chemicals. Thus, GSH in E. coli B is important in maintaining growth in the presence of oxidants but does not affect oxidant lethality.