Radioiodination of synthetic human secretin on its N-terminal histidyl residue was not difficult when a greater amount of Chloramine T and a longer reaction time were employed to achieve better incorporation of 125I. The radioiodinated tracer for an optimal radioimmunoassay required purification. The combination of Sep-pak C18 Cartridge and high performance liquid chromatography for the purification of 125I-secretin in our study revealed that the Sep-pak cartridge was a preliminary step in removing unlabeled radioactive iodide, the reactant, and labeled materials unadsorbed to the cartridge. The eluate eluted from the Sep-pak containing high radioactivity and high immunoreactivity to the antibody were selected for further purification by HPLC which eliminated undesirable radiolabeled substances with lower immunoreactivity. The purified radiolabeled secretin was used in developing a sensitive radioimmunoassay.