Peroral challenge with toxin A from Clostridium difficile induced the formation of antisecretory factor in rats. The animals were given 100 micrograms of the toxin, which was followed by a pronounced diarrhoea and by the appearance of antisecretory factor in the pituitary gland. In electrofocusing, the induced antisecretory factor separated in two peaks (pI 5.4 and 5.0); both fractions showed a lectin-like binding to agarose. The pI 5.4 fraction inhibited cholera toxin as well as toxin A induced fluid secretion, while pI 5.0 inhibited toxin A induced secretion only. Immunohistochemistry showed that an antisecretory factor of pI 5.0 protected the mucosa from the cytotoxic effect of toxin A, but did not affect the binding of toxin A to the intestinal epithelium. Sodium dodecyl-sulphate-polyacrylamide gel electrophoresis of the pI 5.0 protein showed two major fractions to be present, one of molecular weight 60 kDa, the other of 30 kDa, the latter probably being a degradation product of the former.