Rat thyrotropin-releasing hormone prohormone (pro-TRH) is a protein containing five copies of TRH, separated by connecting peptides. We have recently developed radioimmunoassays to synthetic peptides corresponding to prepro-TRH(160-169) and prepro-TRH(178-199). In the present study we have used these assays to investigate the ontogenesis of pro-TRH-derived peptides in the rat pancreas. Reverse-phase HPLC analysis of pancreatic extracts from 2-day-old rats showed the presence of two major immunoreactive peptides exhibiting the same retention time as synthetic prepro-TRH(160-169) and prepro-TRH(178-199), respectively. The concentrations of TRH and pro-TRH cryptic peptides in the rat pancreas rose rapidly after birth, reached a maximum at day 2-4 and decreased gradually afterwards. Streptozotocin treatment of newborn rats induced a marked decrease of TRH (96%), prepro-TRH(160-169) (97%) and prepro-TRH(178-199) content (94%) in pancreatic extracts. These results indicate that the evolution of TRH and pro-TRH-derived peptides follows the same pattern during the postnatal period. Our results also suggest that beta-cells are the only source of pro-TRH-derived peptides in the rat pancreas.