The nucleotide (nt) sequence of the tetracycline resistance (TcR) region (1628 bp) of the Bacillus plasmid pTHT15 was determined. A single open reading frame (ORF), encoding a 458 amino acid (aa) 50-kDal protein (TET), is present after a GTG initiation codon preceded by a ribosome-binding site (RBS-2). The transcriptional start point, at a position 120 nt upstream from the GTG codon was determined by S1 mapping. This upstream region contains a short ORF (30 aa) which is preceded by RBS-1. The presence of three inverted repeats, which can form two different conformations of the mRNA very similar to those of the control region of the macrolide-lincosamide streptogramine B resistance gene of pE194 [Horinouchi and Weisblum, Proc. Natl. Acad. Sci. USA 77 (1980) 7079-7083; Gryczan et al., Nucl. Acids Res. 8 (1980) 6081-6097; Shivakumar et al., Proc. Natl. Acad. Sci. USA 77 (1980) 3903-3907], suggests that the TcR gene is regulated by a translational attenuation mechanism. A Rho-independent transcriptional terminator structure is present immediately after the translational stop codon (TAA) of the TET protein. Comparison of the TET protein with the staphylococcal TcR proteins of pT181 revealed considerable homology.