Fructose-1,6-bisphosphatase (FBPase) plays a crucial role in the regulation of gluconeogenesis. It is a homotetramer with a subunit molecular mass of 37 kDa. Each subunit has one substrate and three metal binding sites. Site-directed mutagenesis were carried out to replace amino acid residues which are thought to play important roles in structure integrity and functioning of the enzyme. Particular emphasis was placed on residues in the substrate and metal binding sites as well as the residues at the C1C2 (C3C4) interface. The mutant and wild-type FBPases were purified to homogeneity and characterized by initial-rate kinetics and circular dichroism spectrometry (CD). There were no discernible differences between the secondary structures of the wild-type and the mutant enzymes on the basis of CD data. Altering G122 to alanine caused a significant decrease in the enzyme's activity and affinity for Mg2+. Most importantly, the mutation caused the total loss of cooperativity for Mg2+. Together with other residues, R276 defines metal site 3 and E280 defines both metal site 1 and 3. Replacement of R276 with methionine caused the total loss of Mg2+ cooperativity and changed the kinetic mechanism from rapid equilibrium random (or steady-state ordered) to rapid equilibrium ordered, in which the substrate adds to the enzyme before Mg2+ and all steps equilibrate rapidly relative to the conversion of the ternary complex. In addition, the mutation abolished the inhibition effects of monovalent cations with the enzyme. When E280 was mutated to glutamine, the enzyme's activity was decreased over 1000-fold, and the mutation changed K+ to a noncompetitive inhibitor with respect to Mg2+. K274, Y264, R243, Y244, and N212 are involved in substrate binding; S123, D127, C128, and residues Y258 and R243 from adjacent chain are located at the C1C2 (C3C4) interface. The mutant FBPases, K274L, Y264F, Y244F, Y258F, R243M, S123A, D127T, and C128G, changed ligand binding affinities;In addition, a kinetic model was developed to account the kinetics and mechanisms of activation and inhibition of FBPase by monovalent cations.