Steady-state absorption and emission, circular dichroism (CD), and time-of-flight secondary-ion-mass-spectroscopic (TOF-SIMS) measurements were performed to study the complexation of tetracycline (TC) and anhydrotetracycline (AHTC) with Mg2+ and Ca2+ ions, respectively, in aqueous solutions at pH 8.02. The results obtained suggest that Ca2+ forms a 1:2 ligand:metal complex with TC via chelation through O10-O11 and O12-O1 and induces thereby the extended conformation A of TC, which is stabilized through hydrogen bonding between the deprotonated dimethylamino nitrogen, N4, and OH12a. pH titrations provide evidence that N4 deprotonates in the presence of a 164-fold molar excess of Ca2+ at approximately pH greater than or equal to 7.7 (c(TC) = 2.1 x 10(-5) M). In contrast to Ca2+, Mg2+ binds to N4-O3 and thereby stabilizes the twisted conformation B of TC. TOF-SIMS measurements indicate that a 1:2 ligand:metal complex is formed in addition to the 1:1 complex. The Mg2+-induced increase in the fluorescence intensity and the observed changes in the absorption spectra provide evidence that the other Mg2+ ion binds to the BCD ring system through the deprotonated Oil. In contrast to TC, which adopts the twisted conformation B in aqueous solution at pH 8.02, AHTC exhibits the extended conformation A due to slightly lower deprotonation constants. In the presence of Mg2+, however, the conformational equilibrium is shifted toward the twisted conformation B due to binding of Mg2+ to N4. TOF-SIMS measurements suggest that a 2:2 ligand:metal complex is formed. AHTC remains in conformation A upon addition of Ca2+; complexation through O10 can be excluded on the basis of absorption spectroscopic data.