A tryptic fragment (b5TR,NR), encompassing residues 2515-2750, was isolated from a low-iodine (0.26 % by mass) bovine thyroglobulin, by limited proteolysis with trypsin and preparative, continuous-elution SDS-PAGE. The fragment was digested with Asp-N endoproteinase and analyzed by reverse-phase HPLC electrospray ionization quadrupole time-of-flight mass spectrometry, revealing the formation of: 1) 3-monoiodotyrosine and dehydroalanine from Tyr2522; 3-monoiodotyrosine from Tyr2555 and Tyr2569; 3-monoiodotyrosine and 3,5-diiodotyrosine from Tyr2748. The data presented document, by direct mass spectrometric identifications, efficient iodophenoxyl ring transfer from monoiodinated hormonogenic donor Tyr2522 and efficient mono- and diiodination of hormonogenic acceptor Tyr2748, under conditions which permitted only limited iodination of Tyr2555 and Tyr2569, in low-iodine bovine thyroglobulin. The present study thereby provides: 1) a rationale for the preferential synthesis of T3 at the carboxy-terminal end of thyroglobulin, at low iodination level; 2) confirmation for the presence of an interspecifically conserved hormonogenic donor site in the carboxy-terminal domain of thyroglobulin; 3) solution for a previous uncertainty, concerning the precise location of such donor site in bovine thyroglobulin.