Protection against intestinal secretion induced by cholera toxin (CT) was studied in adult and suckling rats. Peroral CT treatment of lactating females protected their suckling offspring against diarrhea. This milk contained a protective antisecretory factor (ASF) as shown by passive peroral or intravenous transfer of the milk to adult, nontreated recipients in which the cholera response was tested in ligated jejunal loops. Bile from CT-treated rats also contained ASF, which was still present 100 days after the last treatment. Although milk and bile from untreated rats contained no ASF, bile from very old untreated rats did. Chemical characterization showed that ASF in bile and milk both had isoelectric points of about 5.0 and molecular weights of about 25,000. These data are similar to those previously obtained for ASF isolated from porcine pituitary glands, and show that the antisecretory activity is unrelated to immunoglobulins.