We demonstrate that two enzymes, soluble unspecific pyroglutamyl peptidase I and prolyl endopeptidase, able to degrade thyrotropin-releasing hormone (TRH) in vitro were present in pancreas at the early stage of rat development. Specific particulate pyroglutamyl peptidase II remained undetectable during ontogenesis. Pyroglutamyl peptidase I specific activity increased until day 3 and decreased after day 5. Furthermore, prolyl endopeptidase specific activity rose slightly to a peak on postnatal day 20. A good correlation between immunoreactive TRH and deaminated TRH (TRH-OH) was found in the 1st wk after birth. However, His-Pro diketopiperazine (DKP) levels were stable and low during development. We show that hot acidic extraction conditions could artefactually generate His-Pro DKP. In vivo, active site-directed inhibitors of pyroglutamyl peptidase I and prolyl endopeptidase enzymes do not show any TRH-deamidating and/or pyroglutamyl peptidase I pathways in neonatal rat pancreas. The data suggest that these two enzymes are not involved in intra- or extracellular control of TRH levels in neonatal rat pancreas and that pancreatic TRH content appears to be principally regulated by biosynthetic steps. Nevertheless, low levels of endogenous His-Pro DKP and TRH-OH identified in neonatal rat pancreas suggest that TRH or TRH-like peptides may be metabolized in this tissue in intact rats, albeit at low rates.