Previous work has established that the E. coli htrA gene product is essential for bacterial survival at temperatures above 42 degrees. We have sequenced the htrA gene region and found an open reading frame (ORF) coding for a protein of 491 amino acids with a calculated molecular weight of 51,163 daltons. This molecular weight corresponds well with that seen following electrophoresis on SDS-polyacrylamide gels. This protein has an amino-terminal sequence typical for a leader peptide and undergoes post-translational modification by cleavage of an amino-terminal portion. The insertional mutations which affect the function of the htrA gene map inside this ORF. The levels of htrA mRNA increase rapidly and transiently upon heat shock in a manner independent of the rpoH gene, which encodes the sigma 32 RNA polymerase subunit and is known to regulate transcription of typical heat shock genes. Using S1 mapping and RNA primer extension, we have identified the htrA promoter and found that it is similar to the P3 promoter of the rpoH gene. The P3 promoter is especially active at high temperatures and is recognized by a recently identified transcriptional factor, sigma E.