The mechanism of action of folylpolyglutamate synthetase from Corynebacterium sp. was investigated using tetrahydrofolate, MgATP, and glutamate as the substrates. Initial velocity, product inhibition, and competitive inhibition studies were consistent with an Ordered Ter Ter mechanism with MgATP binding first to the enzyme, tetrahydrofolate second, and glutamate last. The order of dissociation from the enzyme was ADP, folate, and Pi. This mechanism precludes the sequential addition of glutamate moieties to enzyme-bound folate. The Michaelis constants for (dl)-tetrahydrofolate, MgATP, and glutamate were 2,1 muM, 18 MUM, and 160 muM, respectively. Beta, gamma-Methylene-ATP was a very effective inhibitor of the reaction with an affinity for the enzyme 1 order of magnitude greater than that of ATP.