A previous study of the site of metal binding in tetracycline has been extended to several derivatives of tetracycline in an effort to determine which specific functional groups are involved in binding to metal ions in DMSO solution and to explore relationships between antibacterial activity and metal binding characteristics. Proton NMR experiments using paramagnetic and diamagnetic lanthanide series ions as binding site probes indicate that the ring A tricarbonylmethane group is the binding site for tetracycline, 5-hydroxytetracycline, 4-epitetracycline and tetracyclinemethiodide in DMSO. No. NMR evidence for metal binding is found for 4-dedimethylaminotetracycline. Binding to Mg²⁺ is also investigated by NMR for several of these compounds, and a discussion of conformational preferences of tetracycline derivatives in DMSO is presented.