The peptide guanylin, recently isolated from the intestine, and localized to cells of the gut mucosa, is involved in electrolyte/water transport in the intestinal epithelium by means of a paracrine mode of regulation. Since high amounts of this peptide are present also in the systemic circulation, we investigated the adrenal gland as a potential endocrine source of guanylin. Using a reverse transcriptase-polymerase chain reaction and hybridization with an internal oligonucleotide designed for rat guanylin, 514-bp signals were obtained in intestinal tissue and adrenal gland. Successive analyses of extracts from intestine and adrenal gland by HPLC, western blotting, and radioimmunoassay revealed the presence of the same high-molecular mass (about 12.4 kDa) guanylin that corresponds to the mass of the guanylin prohormone. About 60 fmol/ml of circulating immunoreactive guanylin was determined in plasma. Localization studies with antisera directed against different epitopes of guanylin revealed that, in the adrenal gland, guanylin immunoreactivity is restricted to the medulla, where it is mainly confined to norepinephrine chromogranin A-containing cells. On the ultrastructural level, guanylin immunoreactivity was exclusively located to secretory granules of chromaffin cells. The present data indicate that, in addition to entero-endocrine cells, the adrenal medulla represents a further source of guanylin. Thus, an endocrine mode of function of guanylin may accrue to its hitherto evidenced paracrine action in fluid transport in the intestinal epithelium. Furthermore guanylin may be considered as a neurohormonal peptide.