Guanylin and uroguanylin are intestinal peptides that stimulate guanylate cyclase C and cause chloride secretion. These peptides show topological instability due to two disulfide bonds. The disulfide bonds were reduced and S-carboxymethylated to cleave the bonds and obtain stable and sole derivatives. We established a new and reliable RIA system for the stable derivatives from both peptides. With the use of this system, the response of the peptides to salt loading of the rat small intestine was evaluated. The lumen of the small intestines of Sprague-Dawley rats was perfused in vivo with Krebs-Ringer solution containing different concentrations of salt or mannitol. Mature guanylin, proguanylin, and mature uroguanylin were found in the perfusate in the basal state. The highest salt loading (200 mM NaCl for 20 min) increased the guanylin secretion about threefold (1.9 ± 0.2 vs. 5.4 ± 0.5 pmol/min), with the effect lasting for 60 min. The uroguanylin secretion was less affected. Hyperosmolar mannitol also caused a significant but smaller increase of guanylin secretion. Increased guanylin could lead to increased salt and water secretion of the intestine; thus members of the guanylin family have potential roles in the regulation of water and salt metabolism in the small intestine.