At moderate iodination levels (20 iodine atoms/mol) human thyroglobulin (hTg) produces after reduction a hormone-rich peptide of 26 kDa which contains the preferential hormonogenic 'acceptor' tyrosine (Tyr 5) of the protein. The site of cleavage of the hTg chain was demonstrated by analysis of the 26 kDa tryptic hydrolysis products. It consistently yielded the peptide Gln-82-Val-129 which consequently made it possible to localize the hTg chain cleavage at tyrosine residue 130. Evidence for tyrosine involvement in hTg cleavage during thyroid hormone formation supports the hypothesis that peptide bond cleavage would occur at the 'donor' tyrosine residue and suggests that tyrosine 130 would be the donor site reacting with the major hormone-forming acceptor site (Tyr 5) of hTg.