Our previous studies showed that sulfated tyrosines (Tyr-S) are involved in thyroid hormone synthesis and that Tyr₅, the main hormonogenic site of thyroglobulin (Tg), is sulfated. In the present paper we studied the role of Tyr-S in the formation and activity of the peroxidase-Tg complex. Results show that non-iodinated ³⁵SO₃-Tg specifically binds (Kd = 1.758 µM) to immobilized lactoperoxidase (LPO) via Tyr-S linkage by using saturation binding and competition experiments. We found that NIFEY-S, a 15 amino acid peptide corresponding to the NH₂-end sequence of Tg and containing the hormonogenic acceptor Tyr₅-S, was a better competitor than cholecystokinin (CCK8-S) and Tyr-S. ³⁵SO₃-Tg, iodinated without peroxidase, bound to LPO with a Kd (1.668 µM) similar to that of non-iodinated Tg, suggesting that 1) its binding occurs via Tyr-S linkage, 2) Tyr-S requires peroxidase to be iodinated whereas non-sulfated Tyr does not. Iodination of NIFEY-S with [¹²⁵I]iodide showed that Tyr₅-S iodination increased with LPO concentration, while iodination of a non-sulfated peptide containing the donor Tyr₁₃₀ was barely dependent on LPO concentration. Enzymatic hydrolysis of iodinated Tg or NIFEY-S showed that the amounts of sulfated iodotyrosines also depended on LPO amount. Sulfated iodotyrosines were detectable in the enzyme-substrate complex, suggesting they have a short life before the coupling reaction occurs. Our data suggest that after Tyr-S binding to peroxidase where it is iodinated, the sulfate group is removed, releasing an iodo-phenoxy anion available for coupling with an iodotyrosine donor.