Guanylin is a 15-amino acid peptide that acts on intestinal guanylate cyclase, thereby regulating intestinal fluid and electrolyte transport through the second messenger, cyclic GMP. Using synthetic rat guanylin, we prepared an antiserum that recognizes human and rat guanylin equally on a molar basis and developed a sensitive radioimmunoassay (RIA). The major endogenous guanylin molecule in human intestine and plasma is 10-kDa proguanylin, 15-amino acid guanylin being a minor component. Human guanylin is distributed widely from the duodenum to colon, the highest contents being in the ileum and proximal colon. The plasma concentration of immunoreactive guanylin in the normal individuals tested was 31.2 ± 3.0 fmol/ml (mean ± SE) and that in patients with chronic renal failure who were undergoing hemodialysis 7,924 ± 2,140 fmol/ml. The RIA we established is a promising tool for clarifying the physiological functions and pathophysiological significance of guanylin in water and electrolyte homeostasis.