Thyroxine (T₄) is formed by coupling of iodinated tyrosine residues within thyroglobulin (TG). In mature TG, some iodinated tyrosine residues are involved preferentially in T₄ formation. In order to investigate the specific role of various tyrosine residues in T₄ formation, N-terminal TG fragments with mutated tyrosine residues were constructed. An N-terminal TG fragment 198 amino acids in size and containing seven tyrosine residues at amino acid positions 5, 29, 89, 97, 107, 130 and 192 was expressed in a baculovirus system. Using site-directed mutagenesis, eight mutant TG fragments were constructed in which different tyrosine residues were replaced by phenylalanine. In the first four TG mutants, one single tyrosine residue (5, 89, 97 or 130) was mutated. In the mutant Y(5,89,97,130)F all of these four tyrosine residues were replaced. The sixth mutant Y(29,89,107,130,192)F contained only tyrosine residues 5 and 97 and the seventh (Y(29,89,97,192)F) contained only tyrosine residues 5, 107 and 130. A TG fragment (Y(5,29,89,97,107,130,192)F) in which all tyrosine residues were replaced by phenylalanine was used as a negative control. After in vitro iodination with lactoperoxidase, specific T₄ formation was established in the non-mutated wild-type N-terminal TG fragment. In general the T₄ formation in the mutant TG constructs decreased when the total number of tyrosine residues in the 198 amino acid fragment decreased, except fragment Y(29,89,97,192) containing three tyrosine residues, two of them being 5 and 130. Although the rate of T₄ formation in this mutated N-terminal TG fragment was lower, the ultimate T₄ generation was the same as in the wild-type fragment. This indicates that a preferential involvement of tyrosines 5 and 130 in thyroid hormonogenesis may exist.