AbstractIodination of the conserved 2‐tyrosine (Tyr2) residue in the pressin and tocin rings of arginine‐ or lysine‐vasopressin (AVP or LVP), and oxytocin, respectively, impairs binding to their respective receptors. Synthetic antagonists that have their Tyr2 either replaced by another amino acid or irreversibly blocked by an O‐methyl or O‐ethyl ether, but have, instead, an iodinatable phenol moiety outside the pressin/tocin ring, are used for radiolabeling. We explored another approach to avoid iodinating Tyr2 by capping this residue with a reversible O‐acetyl group, incorporated during peptide synthesis. The O‐acetyl‐Tyr2 LVP peptide, with a free iodinatable tyrosine attached to the ε‐amine of 8‐lysine, is iodinated at a neutral pH and purified by reverse‐phase high‐pressure liquid chromatography (HPLC) at an acidic pH, conditions under which the O‐acetyl groups are stable. Deacetylation with hydroxylamine is selective, and leaves intact the disulfide bridge. The marked shortening of the HPLC retention time after deblocking produces a chemically homogeneous label, iodinated exclusively on the free tyrosine residue attached to the ε‐amine of LVP. Hitherto, this 125I labeled vasopressin agonist could be obtained only in low yield, via conjugation labeling with iodinated N‐t‐Boc‐tyrosine succinimidyl ester. This fully reversible tyrosine protection strategy does not require special equipment, and retains the conserved Tyr2, typical of vasopressin and oxytocin agonists. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd.