Several studies have suggested that the metabolism of one-carbon compounds may have a special role in the function of the exocrine pancreas. An amino acid-defined diet was used to produce folate deficiency in a group of male rats. These rats were compared with a group of rats pair-fed the same diet supplemented with adequate folate and with a third group fed the folate-supplemented diet with ad libitum access. Pancreatic folate concentrations were already severely depleted after 4 wk of feeding the deficient diet (0.95 +/- 0.10, 5.81 +/- 0.29 and 4.58 +/- 0.30 nmol/g for the deficient, pair-fed control and ad libitum-fed control groups, respectively). The level of folate present in the pancreas of nondeficient animals was second only to that reported for liver. Urinary amylase excretion by animals in the deficient group was higher than that by the other groups (245.5 +/- 21.9, compared with 181.9 +/- 14.5 and 195.3 +/- 10.9 units/mg creatinine for the deficient, pair-fed control and ad libitum-fed control groups, respectively) after 4 wk. The ratio of S-adenosylmethionine to S-adenosylhomocysteine was 18.6 +/- 1.6 and 14.5 +/- 1.0 after 4 wk for the ad libitum-fed control and pair-fed control groups, respectively, but was significantly lower at 6.3 +/- 1.1 for the deficient group. These results indicate a profound effect of folate deficiency upon methyl group metabolism of the pancreas and suggest that this may result in decreased pancreatic function.