Previous studies have suggested that the metabolism of methyl groups is an important factor in the function of the exocrine pancreas. Ethionine, an inhibitor of cellular methylation reactions, produces hemorrhagic pancreatitis when administered to mice fed a choline-deficient diet. Glycine N-methyltransferase, an enzyme which regulates the ratio of S-adenosylmethionine to S-adenosylhomocysteine, is particularly abundant in the exocrine pancreas. Since de novo synthesis of methyl groups requires the participation of folate coenzymes, we investigated the effect of folate deficiency on pancreatic exocrine function. Rats were fed an amino acid-defined folate-deficient diet or the same diet supplemented with folate ad libitum. A third group received the folate supplemented diet pair-fed to the deficient group. After 3 and 5 wk, pancreatic amylase secretion was measured in perfused duodenal segments of anesthetized animals before and after cholecystokinin injection. Pancreatic secretion was significantly reduced in the deficient group compared with the pair-fed control group after 5 wk. These results indicate that severe folate deficiency impairs pancreatic exocrine function.