Isolated rat pancreatic islet extracts were submitted to reverse phase high pressure chromatography (HPLC) and assayed for thyrotrophin releasing factor (TRF), glucagon and insulin in radioimmunoassays (RIA). Our anti-TRF serum was prepared by immunizing rabbits with TRF conjugated to bovine thyroglobulin (bTG) by bisdiazobenzidine. Anti-glucagon and antiinsulin were from a commercial source (Novo, Denmark). The concentrations of TRF, glucagon and insulin were 0.05 +/- 0.02, 0.06 +/- 0.02 and 1.0 +/- 0.2 pmol/islet, respectively. Formalin-fixed pancreatic sections were stained for TRF, glucagon and insulin by peroxidase, antiperoxidase (PAP) complex method. When the adjacent sections were stained for glucagon or insulin, it was observed that TRF and glucagon-specific peroxidase reactions were confined to the marginal islet cells, but insulin reactions to the central cells. The TRF-specific peroxidase reaction was clearly reduced when the anti-TRF serum was unchanged when the antiserum was pre-incubated with synthetic TRF. The present HPLC results suggest that the islets contained TRF. Immunocytochemical studies show that the TRF-immunoreactive material, either synthesized or bound, is localized in the marginal islet cells.