We studied the in vitro synthesis of thyrotropin releasing hormone (TRH) by pancreatic cells, using [14C]histidine incorporation and radioimmunoassay. Neonatal pancreases were incubated in Krebs-Ringer-bicarbonate buffer, pH 7.4, at 37 degrees C, under 95% O2-5% CO2. [14C]histidine was added and its incorporation into the tissue proteins was followed up to 4 hours. The methanolic pancreatic extracts obtained were subjected to Sephadex G-200 chromatography. Two peaks of [14C] radioactivity were eluted, one small (20% of total radioactivity) with the void volume and one large in a position identical to synthetic TRH. Both radioactive peaks corresponded to immunoreactive TRH peaks present in the same samples. In pulse-chase experiments, excess cold histidine was used and samples taken showed again similar radioactive peaks with an initial increase and a later decrease in the size of the small peak. We conclude that the neonatal rat pancreas actively synthesizes TRH and/or a higher molecular weight TRH-like protein, which may represent a TRH precursor.