Abstract The antisecretory factors (ASF) are hormone-like proteins which inhibit cholera toxin-induced intestinal hypersecretion. Although ASF concentrations in young control rats were low, those in old control rats and toxin-treated rats were high. Toxin-treated rats had 200 ED 50 units/g wet weight of ASF in the pituitary gland, while their intestinal mucosa, bile and milk contained 3, 0.5 and 0.5 units/g. In adult man and in 8–9-month-old pig the pituitary level was about 20 units/g. The isoelectric points of ASF from pig and rat were 4.8 and 5.0, respectively, while the molecular size as determined by gel filtration on Bio-Gel P-150 was the same in both cases ( K av 0.43). The molecular weight as determined by SDS-polyacrylamide gel electrophoresis was 60 000 for ASF from porcine pituitary gland. One ED 50 unit of the purified porcine ASF corresponded to about 10 −13 mol (1–5 ng) of protein. There were two different ASF from human pituitary gland: (i) p I 5.2, K av 0.43; and (ii) p I 4.5, K av 0.6. Since antibodies against porcine ASF failed to neutralize the latter protein, it may be unrelated to porcine ASF; the human p I 5.2-protein and rat ASF were both neutralized, but less effectively than was porcine ASF. All the ASF molecules attached to agarose gel, from which they dissociated again in methyl α- d-glucose: porcine and rat ASF were eluted at 0.3–0.9 M methyl α- d-glucose, human p I 5.2-ASF at 0.1–0.9 M, and human p I 4.5-ASF at 0.1–1.5 M methyl α- d-glucose.