Immunoreactive TRH-OH is present at low concentrations in acid extracts from 2- days old rat pancreas. The sequential treatment of these extracts with trypsin and carboxypeptidase A is followed by a three- and ten-fold increase in TRH-OH IR respectively. The molecular weight of the protein that gives rise to TRH-OH after enzymatic treatment ranges between 30000 and 40000 daltons. The appearance of TRH-OH in the tryptic digest suggests that TRH-OH is the COOH-terminal sequence of this protein. These results are the first evidence that TRH biosynthesis occurs through a large molecule precursor. However, this is an indirect demonstration since TRH cannot be generated under these conditions due to the lack of enzymatic amidation activity.