The location of Tn10 genes encoding tetracycline resistance and its regulation was determined by analyzing the properties of recombinant plasmids carrying partial HpaI digestion products of lambda::Tn10 transducing phage deoxyribonucleic acid. Within a 2,700-base pair region are encoded tetracycline resistance, the structural gene (tet) for a tetracycline-inducible polypeptide, and the regulatory elements for the induction of both the resistance phenotype and the polypeptide. Fusion of different sequences to an HpaI site in the tet gene alters the molecular weight and stability of the polypeptide as well as the tetracycline resistance phenotype of strains producing fusion polypeptides. These results indicate the orientation of the tet gene and support the conclusion that the tet polypeptie is required for tetracycline resistance. A HincII cleavage site immediately upstream from the tet gene is protected by ribonucleic acid polymerase, but only the absence of ribonucleotide triphosphates. The possibility that tet transcription is initiated at this site is discussed.