We used the Tn10-encoded Tet repressor, which has a highly specific binding capacity for tetracycline, to probe contacts between the drug and protein by chemical interference studies of the antibiotic. For that purpose, the equilibrium association constants of modified tetracyclines with the Tet repressor and Mg2+ cations were determined quantitatively. The results confirm the previous notion that Mg2+ probably binds with the oxygens at positions 11 and 12 and is absolutely required for protein-drug recognition. Modifications were introduced at positions seven, six, five, and four of the drug, and anhydrotetracycline was also studied. Substitutions or eliminations of functions at these positions influenced binding to the Tet repressor up to 35-fold. The introduction of an azido function at position seven in 7-azidotetracycline and epimerization of the substituents at position four in 4-epitetracycline lead to a 2- or 25-fold reduction, respectively, of Tet repressor affinity in those compounds. Anhydrotetracycline bound about 35-fold more strongly than tetracycline did, indicating that the oxygen at position 11 may be involved in Tet repressor recognition. This increased binding is in contrast to the lower antibiotic activity of anhydrotetracycline and indicates that the Tet repressor and ribosomes recognize the drug differently.