The kinetic mechanisms of Escherichia coli phosphofructokinase-2 (Pfk-2) and of the mutant enzyme Pfk-2 were investigated. Initial velocity studies showed that both enzymes have a sequential kinetic mechanism, indicating that both substrates must bind to the enzyme before any products are released. For Pfk-2, the product inhibition kinetics was as follows: fructose-1,6-P2 was a competitive inhibitor versus fructose-6-P at two ATP concentrations (0.1 and 0.4 mM), and noncompetitive versus ATP. The other product inhibition patterns, ADP versus either ATP or fructose-6-P were noncompetitive. Dead-end inhibition studies with an ATP analogue, adenylyl imidodiphosphate, showed uncompetitive inhibition when fructose-6-P was the varied substrate. For Pfk-2, the product inhibition studies revealed that ADP was a competitive inhibitor versus ATP at two fructose-6-P concentrations (0.05 and 0.5 mM), and noncompetitive versus fructose-6-P. The other product, fructose-1, 6-P2, showed noncompetitive inhibition versus both substrates, ATP and fructose-6-P. Sorbitol-6-P, a dead-end inhibitor, exhibited competitive inhibition versus fructose-6-P and uncompetitive versus ATP. These results are in accordance with an Ordered Bi Bi reaction mechanism for both enzymes. In the case of Pfk-2, fructose-6-P would be the first substrate to bind to the enzyme, and fructose-1,6-P2 the last product to be released. For Pfk-2, ATP would be the first substrate to bind to the enzyme, and APD the last product to be released.