The E. coli heat shock transcription factor, σ³², is a very unstable protein, can be degraded by several ATP-dependent proteases, and its stability is subjected to dynamic regulation by molecular chaperones, such as DnaK and DnaJ. To gain new insights into the substrate recognition mechanisms of these proteolytic machinaries including chaperones, we have isolated several σ³² mutants with markedly increased stability in vivo. The mutants obtained were found to contain one or more amino acid changes in region 2.1, one of the highly conserved regions among bacterial s factors. One mutant, with a half-life of more than 10 min (>10-fold higher than the wild type), contained two amino acid changes (L47Q and L55Q) in this region, whereas two other mutants, A50S and I54A, exhibited half-lives of about 5 and 10 min, respectively. Besides longer half-life, they all showed higher transcriptional activity and produced several-fold higher levels of heat shock proteins as comapred to the wild type. These results suggest the interesting possibility that part of region 2.1 of σ³² protein is involved in interaction with some proteolytic machinery, and that certain amino acid residues of this region are required for modulating activity as well as metabolic stability of σ³².