The Escherichia coli cell permeability-cell separation gene envA and the region immediately downstream were sequenced. The envA gene consisted of 305 codons which encoded a 34-kilodalton polypeptide that lacked a signal sequence and hydrophobic membrane-spanning regions. The envA1 mutation was determined to be a missense mutation in codon 19 resulting in a change in the amino acid sequence from histidine to tyrosine. Located 299 base pairs downstream of the envA gene was an unidentified open reading frame consisting of 147 codons. This open reading frame was followed by an additional open reading frame starting 59 base pairs further downstream and corresponded to the secA gene. A transcription terminator was located just downstream of envA on a fragment that contained a sequence corresponding to a typical rho-independent terminator. Transcription of envA and the upstream fts genes terminated at this terminator and was probably uncoupled from the downstream genes, including secA. Gene disruption experiments indicated that the envA gene was an essential gene.