Glycine N-methyltransferase (GNMT) regulates the ratio of S-adenosylmethionine to S-adenosylhomocysteine. It is very abundant in liver cytosol and earlier studies have shown it to be present in high concentrations in the pancreas. We have previously reported that liver GNMT is allosterically inhibited by 5-methyltetrahydrofolate pentaglutamate (5-CH3-H4PteGlu5), and proposed that this represents a metabolic control mechanism which links the de novo synthesis of methyl groups to the methylating ability of the liver. We now report that pancreatic GNMT also contains bound folate in vivo. Purified pancreatic GNMT is inhibited by reduced folate polyglutamates in vitro. The KI for the synthetic (R,S)5-CH3-H4PteGlu5 is 2.4 x 10(-7) M. The natural (S) form of 5-CH3-H4PteGlu5 is tightly bound and has a Kd of 1.3 x 10(-7) M. One mole is bound per enzyme tetramer. These studies suggest that GNMT is important in the regulation of methyl group metabolism in the pancreas as well as in the liver.