The kefC gene of Escherichia coli encodes a potassium efflux system that is gated by glutathione (GSH) and by GSH adducts. Independently isolated kefC mutations that result in spontaneous activation of the efflux system have been analyzed. Three mutations affect residues located adjacent to the conserved Rossman fold in the carboxyl-terminal domain. Two mutations lie in a sequence predicted to form a cytoplasmically located loop in the membrane domain of KefC. All of the mutants retain normal regulation by the YabF protein and by GSH adducts. A mutation in the Rossman fold, R416S, alters the normal regulation of KefC by GSH. In contrast to the wild-type protein, which is inactive in the presence of GSH, the R416S mutant is only active in the presence of GSH or its analogue, ophthalmic acid. Other mutations in this region or elsewhere in the protein have their spontaneous activity augmented by depletion of the GSH pool. These data identify a specific role for the carboxyl-terminal domain of KefC in regulating KefC activity and are discussed in the light of recent data that suggest that GSH adducts can bind within a Rossman fold.