We have determined the transcriptional organization of the Escherichia coli dnaX gene, the structural gene for both the gamma and tau subunits of DNA polymerase III holoenzyme. By S1 nuclease protection and primer extension mapping of transcripts encoding the dnaX products, one primary promoter of dnaX has been identified that initiates transcription 37 nucleotides upstream from the first codon. dnaX resides in an operon with two recently sequenced genes, orf12, encoding an unidentified product, and recR, the structural gene for a protein involved in the recF pathway of recombination. Under conditions of balanced growth, a very small amount of transcription from the upstream apt promoter (less than 5%) contributes to the expression of tau and gamma, too low for apt to be considered to be on an operon with dnaX, orf12, and recR are transcribed from an independent promoter as well as from the dnaX promoter, providing a mechanism for orf12 and recR to be regulated independent of dnaX. Transcription of the dnaX-orf12-recR operon is terminated upstream from the previously characterized heat shock gene htpG. The dnaX and orf12-recR promoters, cloned into a promoter detection vector, efficiently direct the expression of the downstream reporter gene, lacZ. These results extend our knowledge of the genetic and transcriptional organization of this region of the E. coli chromosome. The transcriptional organization has been defined as follows: apt, dnaX-orf12-recR, htpG. All of these genes are transcribed in the clockwise direction and only dnaX, orf12 and recR are contained in the dnaX operon.