Guanylin, an activator of the guanylyl cyclase C receptor in the apical membrane of intestinal epithelium, modulates intestinal fluid and electrolyte transport. The bioactive 15-amino acid peptide originally isolated from rat intestine represents the C-terminal part of a longer, 115-residue prepropeptide. The aim of the present study was to characterize the direction and molecular form in which guanylin is secreted from the colonic mucosa, as well as the mechanisms that trigger its secretion. Isolated rat colonic mucosa was mounted in Ussing chambers, allowing the separate determination of apical and basolateral release. After HPLC purification, two different molecular forms of guanylin were identified in the apical incubation media by combining a bioassay for guanylyl cyclase C activation, a specific guanylin enzyme-linked immunosorbent assay and mass spectrometry, as well as sequence analysis: a bioactive form coeluting with synthetic 15-residue guanylin and the 94-residue propeptide, guanylin-22–115. The basal concentration of proguanylin at the apical side of epithelia was about 15-fold higher, compared with that of the small, bioactive peptide. In the basolateral incubation media, no proguanylin and only very low amounts of bioactive guanylin were detected. Incubation with carbachol led to a significant increase of about 7-fold in the release of proguanylin to both sides of the isolated epithelia. On the apical side, a concomitant increase of the small, bioactive peptide was observed; whereas, on the basolateral side, its concentration remained unchanged. Vasoactive intestinal peptide or the NO-donor S-nitroso-N-acetylpenicillamine did not affect guanylin secretion. Our results suggest that, in the intestine, guanylin is secreted mainly to the luminal side of the epithelium. The peptide is released as a 94-residue propeptide, which is then processed to a smaller, bioactive form (luminocrine secretion). Carbachol stimulates the release of proguanylin to both sides of the intestinal mucosa, but a parallel increase in the bioactive C-terminal derivative only occurs on the apical side. In vivo, the basolateral release could be a source of circulating proguanylin, which might be processed proteolytically to the active peptide in distant target tissues (endocrine secretion).