The nucleotide sequence of the 5′ upstream region of the Escherichia coli rpoS gene was determined and analyzed. At least four promoters responsible for rpoS transcription were identified, and designated P1, P2, P3 andP4, P1 being furthest from the upstream. Using lacZ fusion genes, the P2 promoter was found to be the strongest of the four. All of these promoters are regulated similarly, and their activity is enhanced 2 to 3-fold in stationary phase. P1 and P2 transcription start sites were determined by primer extension analyses. The P2 promoter region shows similarity to the consensus σ⁷⁰-type promoter sequence, and was recognized by both Eσ⁷⁰ and Eσ³⁸ holoenzymes in vitro. The mRNA transcribed from the most distal promoter, P1, appears to include another open reading frame (orf-281), indicating that the two open reading frames comprise an operon. The rpoS gene product (σ³⁸) was rapidly degraded after addition of chloramphenicol to cultures in the exponential, but not the stationary phase. This strongly suggests that posttranslational regulation is involved in the control of rpoS expression.