Dihydrofolate synthetase (EC 6.3.2.12) from N. gonorrhoeae was isolated and enzyme characteristics were determined. The purified enzyme was found quite stable when stored at -60 degrees C. About 50% of the enzyme activity was destroyed within 6 weeks when kept at 4 degrees C. Maximum velocity was observed at pH 9.3. The enzyme required a monovalent cation, K+ or NH4+, and divalent cation, Mg2+ or Mn2+, for its function. ATP at 5 mM concentration gave maximum activity. Km values for dihydropteroate and L-glutamate at pH 9.3 were 3.5 X 10(-5) M and 6.5 X 10(-4) M, respectively. Patterns of product inhibition by dihydrofolate were found to be non-competitive with respect to dihydropteroate, having a Ki value of 5.1 +/- 0.8 X 10(-4) M, and competitive with respect to L-glutamate, having a Ki value of 6.2 X 10(-4) M.