Overproduction of DnaK in Escherichia coli results in a bacteriocidal effect. This effect is most acute in stationary phase cells. A selection scheme was developed to isolate multicopy suppressors from an E. coli plasmid expression library, which overcome the stationary phase toxicity of excess DnaK. Two suppressor plasmids were recovered which contained inserts of 1.85 kb and 2.69 kb, respectively. Rearranged and deleted plasmid derivatives were constructed and used to further localize the suppressors. DNA sequence analysis demonstrated that one suppressor encoded phosphogluconate dehydratase (Edd) while the other suppressor encoded the N-terminal 237 amino acids of DnaK itself (DnaK'). Strains bearing the suppressor plasmids constitutively overproduced proteins with apparent masses of 66 kDa (Edd) and 37 kDa (DnaK') as determined by gel electrophoresis. Western blot analysis using polyclonal antisera specific for either Edd or DnaK confirmed the identity of these overproduced proteins. Suppression of DnaK toxicity was eliminated by the introduction of a + 1 frameshift mutation early in the respective coding regions of either of the two suppressors. These results suggest that suppressor gene translation plays a role in the mechanism of DnaK suppression.