Administration of minocycline (MN), a tetracycline antibiotic, produces a black pigment in the thyroids of humans and several species of experimental animals and antithyroid effects in rodents. We have previously shown that these effects appear to be related to interactions of MN with thyroid peroxidase (TPO), the key enzyme in thyroid hormone synthesis. In the present study, the mechanisms for inhibition of TPO-catalyzed iodination and coupling reactions by MN were investigated. MN was stable in the presence of TPO and H2O2, but adding iodide or a phenolic cosubstrate caused rapid conversion to several products. TPO-dependent product formation, characterized by on-line LC-APCI/MS and 1H-NMR, involved oxidative elimination to form the corresponding benzoquinone with subsequent dehydrogenation at the aliphatic 4-(dimethylamino) group. Addition of thiol-containing polymers (bovine serum albumin or thiol-agarose chromatographic beads) had a minimal effect on MN oxidation by TPO, but substantially reduced product formation and produced concomitant losses in free thiols. Covalent bonding through a thioether linkage of a reactive intermediate, the benzoquinone iminium ion, was inferred from these findings. Iodide- and phenolic cosubstrate-dependent oxidation of tetracycline to demethylated and dehydrogenated products was also observed, although at a slower rate than MN. The products and kinetics observed with MN were consistent with oxidation of MN by either the enzymatic iodinating species formed by reaction of TPO compound I with iodide or phenoxyl radicals/cations generated by TPO-mediated oxidation of a phenolic cosubstrate. The proposed reaction mechanism is consistent with alternate substrate inhibition of TPO-catalyzed iodination of tyrosyl residues in thyroglobulin (Tg) by MN, as previously reported. Furthermore, the observed phenoxyl radical-mediated oxidation of MN is consistent with its previously reported potent inhibition of the coupling of hormonogenic iodotyrosine residues in Tg in the reaction that forms thyroid hormones. The proposed reaction mechanism also implicates a reactive benzoquinone iminium ion intermediate that could be important in toxicity of MN.