Most mammalian cells receive exogenous folate from the bloodstream in the form of 5‐methyltetrahydropteroylmonoglutamate (CH3‐H4PteGlu1). Because this folate derivative is a very poor substrate for folylpolyglutamate synthetase, the enzyme that adds glutamyl residues to intracellular folates, CH3‐H4PteGlu1must first be converted to tetrahydropteroylmonoglutamate (H4PteGlu1), 10‐formyltetrahydropteroylmonoglutamate (CHO‐H4PteGlu1), or dihydrofolate (H2folate), which are excellent substrates for folylpolyglutamate synthetase. Polyglutamylation is required both for retention of intracellular folates and for efficacy of folates as substrates for most folate‐dependent enzymes. Two enzymes are known that will react with CH3‐H4PteGlu1in vitro, methylenetetrahydrofolate reductase and methyltetrahydrofolate‐homocysteine methyltransferase (cobalamin‐dependent methionine synthase). These studies were performed to assess the possibility that methylenetetrahydrofolate reductase might catalyze the conversion of CH3‐H4PteGlu1to CH2‐H4PteGlu1. CH2‐H4PteGlu1is readily converted to CHO‐H4PteGlu1by the action of methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase, and these enzyme activities show very little preference for folylpolyglutamate substrates as compared with folylmono‐glutamates. We conclude from in vitro studies of the enzyme that methylenetetrahydrofolate reductase cannot convert CH3‐H4PteGlu1to CH2‐H4PteGlu1under physiological conditions and that uptake and retention of folate will be dependent on methionine synthase activity.—Green, J. M.; Ballou, D. P.; Matthews, R. G. Examination of the role of methylenetetrahydrofolate reductase in incorporation of methyltetrahydrofolate into cellular metabolism.FASEB J.2: 42‐47; 1988.