We have previously established a two-plasmid system in Escherichia coli for identification of promoters recognized by RNA polymerase containing a heterologous σ factor. Attempts to optimize this system for identification of promoters recognized by RNA polymerase containing E. coli extracytoplasmic stress response σ E failed owing to high toxicity of the expressed rpoE. A new system for identification of σ E-cognate promoters was established, and verified using the two known σ E-dependent promoters, rpoEp2 and degPp. Expression of the σ E-encoding rpoE gene was under the control of the AraC-dependent P BAD promoter. A low level of arabinose induced a non-toxic, however, sufficient level of σ E to interact with the core enzyme of RNA polymerase. Such an RNA polymerase holoenzyme recognized both known σ E-dependent promoters, rpoEp2 and degPp, which were cloned in the compatible promoter probe plasmid, upstream of a promoterless lacZα reporter gene. This new system has proved to be useful for identification of E. coli σ E-cognate promoters. Moreover, the system could be used for identification of ECF σ-cognate promoters from other bacteria.