The DNA sequences of the diadenosine tetraphosphatase gene (apaH) and of the flanking regions were determined. Three other genes were identified in the flanking regions: ksgA, apaG and folA encoding, respectively, a 16 S rRNA methyltransferase, an unidentified protein of Mr 13,826 and dihydrofolate reductase, with the order folA-apaH-apaG-ksgA. The apaH gene is thus located between folA and ksgA at 1 min on the Escherichia coli chromosome linkage map and folA is transcribed clockwise, whereas ksgA, apaG and apaH are transcribed in the opposite direction. It was shown that ksgA, apaG and apaH can be expressed from a polycistronic mRNA originating from a promoter (p1) located upstream of ksgA. However, another promoter (p2) was found within the ksgA structural gene. This promoter, active in vivo, can account for p1-independent expression of the two distal cistrons, apaG and apaH. Finally, the effect on diadenosine tetraphosphatase over-production of a frameshift mutation causing premature translational termination of apaG suggests that expression of apaG and apaH is coupled at the translational level.