The antisecretory factor (AF) is a new regulatory protein, produced in the human pituitary gland, which reverses intestinal fluid secretion induced by cholera toxin. We have previously described the cDNA-cloning and characterization of the expressed gene. The aim of this study was to identify the region responsible for the antisecretory activity in the AF-molecule. The recombinant full-length AF has an increased ability to inhibit hypersecretion after treatment with trypsin, indicating that the activity of AF is achieved by smaller peptide fragments. To localize the active region of AF, we expressed truncated forms of the recombinant protein and examined their antisecretory activity against cholera toxin-induced fluid secretion in rat. Nine recombinant AF peptides and four smaller peptides made by solid phase synthesis were tested. Five of the peptides lacked all activity, whereas seven of them were highly active, a dose between 4 and 15 pmol causing a half-maximal inhibition. All the active peptides contained amino acid 36-42 of the AF sequence, whereas none of the inactive peptides contained this sequence. Our results suggest that the site of the antisecretory activity resides in a small region (I)VCHSKTR between position 35 and 42 of the AF molecule.