Tetracycline depends on divalent metal ions for its biological function, but its multiple ionization states, conformations, and tautomers at varying solution conditions complicate its ion-binding equilibria, and the stoichiometry of the biologically relevant Ca2+ or Mg2+ complexes has not been clear. Isothermal titration calorimetry was used in the present work to study Ca2+ and Mg2+ binding to tetracycline. The two metal ions bind with distinct stoichiometries, one Ca2+ per tetracycline and one Mg2+ per two tetracyclines, and with differing dependence on solution conditions, indicating that these two ions bind TC differently. An endothermic process accompanies ion binding that is proposed to reflect conformational changes in tetracycline. The results identify conditions that limit the distribution of species and may facilitate structural study.