We have investigated the properties of the rat liver folylpoly-glutamate synthetase using methotrexate (MTX; 4-NH2-10-CH3-PteGlu) as a substrate. Many characteristics of the synthetase (e.g., the apparent Km values for L-glutamate and ATP, and the optimal concentrations of KCl and 2-mercaptoethanol) are virtually identical whether MTX or tetrahydrofolate is the “folate” substrate. There are, however, several significant differences between the reactions catalyzed with these two substrates. The length of products synthesized from tetrahydrofolate are inversely related to the initial monoglutamate concentration. Low tetrahydrofolate concentrations allow synthesis of longer (n = 3) polyglutamates, up to pentaglut-amate length, while high concentrations lead to predominantly di-glutamate synthesis. However, 4-NH2-10-CH3-PteGlu2 predominates regardless of the initial MTX concentration, under otherwise identical conditions. Also, tetrahydrofolate can be readily converted to pentaglutamate lengths, the same as predominates in rat liver in vivo. In contrast, MTX forms species containing only up to a total of three glutamates, i.e., 4-NH2-10-CH3-PteGlu3. Finally, the ultimate product of synthesis from tetrahydrofolate, H4teGlu5, is a fairly good inhibitor of synthetase activity with either MTX or tetrahydrofolate as the substrate. The ultimate product of MTX synthesis, 4-NH2-10-CH3-PteGlu3, however, is a poor inhibitor of activity with either substrate.