KefC is a glutathione-gated K(+)-efflux system that is widespread in Gram-negative bacteria and which plays a role in the protection of cells from the toxic effects of electrophilic reagents, such as N-ethylmaleimide (NEM). The KefC gene from Escherichia coli has been cloned and the DNA sequenced. A number of kefC mutants that affect K+ retention by the KefC system have been isolated and all retain activation by NEM. Cloned kefC was found to suppress the phenotype of two such mutants kefC121 and kefC103. Analysis of this phenomenon has shown that suppression is specific to the KefC system, but that cloned kefC from Klebsiella and Erwinia can also mediate suppression of the mutant phenotype. Plasmid constructs of the E. coli gene in which expression of the cloned gene was diminished showed induced ability to suppress the mutant phenotype. KefC'-'LacZ hybrid proteins were inserted in the membrane but did not suppress the mutant phenotype. Cloned kefC did not suppress a mutant kefB allele that exhibited a similar phenotype to the kefC121 allele. These data suggest that suppression is unlikely to arise from exclusion of the mutant form of the protein from the membrane. Furthermore, NEM-activated K+ efflux from a strain carrying both the mutant and cloned wild-type alleles was faster than when either allele was present in cells alone, suggesting that both forms of the protein are inserted into the membrane. These data are discussed in terms of a model for the KefC protein in which the protein is composed of one or more identical subunits that interact in the membrane.