Summary Folylpolyglutamate synthetase (FPGS; EC 6.3.2.17), an enzyme in the folate biosynthetic pathway, which catalyzes the conversion of folates to their polyglutamate derivatives, has been purified 2,917-fold from cultured cells of Datura innoxia by a protocol involving fractionation with 40–65% ammonium sulphate, followed by column chromatography on DEAE-cellulose, phenylagarose, Sephadex G-75, AMP agarose, and MTX agarose. This is the first time that affinity chromatography on AMP-octa-agarose and MTX agarose was used in FPGS purification. The Datura FPGS is a monomer with a molecular weight of about 68,000 u (Da). The purified protein requires folate, ATP, Mg2+, and glutamate for activity and has maximum activity at pH 9.5. The best folate substrate was tetrahydrofolate followed by dihydrofolate, folinic acid, 5-methyl tetrahydrofolate, methotrexate and folic acid. ATP was the best nucleotide substrate, with dATP ranked second; GDP, CTP and UTP were all poor substrates. The divalent cation Mg2+ at a concentration of 20 mmol · L−1 (ATP = 5mmol · L−1) stimulated activity most effectively. The monovalent cations, Li+ and Na+, had no significant effect on Datura FPGS activity, while K+ and NH4+ inhibited the activity. The apparent Michaelis constants were, for tetrahydrofolate, 6.8 mmol · L−1; for ATP, 35.7 mmol · L−1; and for L-glutamate, 150 mmol · L−1.