Sensitive radioimmunoassay for secretin was developed by using synthetic preparation of porcine secretin and its related analogs. The secretin-specific antisera with titers ranging 1: 20,000-1 : 150,000 were generated in rabbits against highly purified synthetic secretin. The labeled antigen was prepared by radioiodinating by the chloramine-T method synthetic secretin analog, Nalpha-tyrosylsecretin or [Tyr1]-secretin, both of which were proved to have almost identical immunoreactivities with that of secretin itself. The immunoassay was performed by the double-antibody method using synthetic secretin as standard. The lowest detectable amount of secretin in the present assays was 5-10pg/tube. Human duodenum extract with hot water contained secretin or secretin-like material that shows a parallel displacement curve to the standard in the immunoassay system used. Serum levels of secretin immunoreactivity in man rose up to 250 pg/ml by intraduodenal infusion of HCl and to 800-1,000 pg/ml by i.v. injection of 1 cu/kg of Boots natural secretin.