We have previously examined the genetic organization and regulation of the Tn10 tetracycline-resistance determinant in Escherichia coli K-12. The structural genes for tetA, the Tn10 tetracycline-resistance function, and for tetR, the Tn10 tet repressor, are transcribed in opposite directions from promoters in a regulatory region located between the two structural genes. Expression of both tetA and tetR is induced by tetracycline. Here we report the DNA sequence of the Tn10 tet regulatory region. The locations of the tetA and tetR promoters within this region were defined by S1 nuclease mapping of the 5' ends of in vivo tet RNA. The tetA and tetR promoters overlap; the transcription start points are separated by 36 bp. We propose that two similar regions of dyad symmetry within the Tn10 tet regulatory region are operator sites at which tet repressor binds to tet DNA, thereby inhibiting transcription initiation at the tetA and tetR promoters. The Tn10 tet regulatory region and the pBR322 tet regulatory region show significant DNA sequence homology (53%).