The effect of thiol group modification of rabbit liver fructose-1,6-bisphosphatase by N-ethylmaleimide (NEM) was investigated. The results of these studies suggest that the substrate, fructose 1,6-bisphosphate (Fru-1,6-P2), is almost as effective as the inhibitor, fructose 2,6-bisphosphate (Fru-2,6-P2), in preventing loss of Fru-2,6-P2 inhibition when the enzyme is modified by NEM. What differences exist between the two ligands in their ability to protect against loss of Fru-2,6-P2 inhibition seem to be quantitative rather than qualitative. Our experimental results suggest that Fru-2,6-P2 binds to the enzyme at its active site;The binding site(s) in rabbit liver fructose-1,6-bisphosphatase for the active site binding ligand, fructose 6-phosphate, and the inhibitor, Fru-2,6-P2, have been investigated by using nuclear magnetic resonance spectroscopy (NMR). The distance from a nitroxide spin label to the bound ligands and the distance from the structural metal site to the bound ligands are about the same within experimental error. These data indicate that the two ligands probably bind at the active site in the rabbit liver enzyme;The interaction of the ligands with fructose 1,6-bisphosphatase were investigated by proton NMR. Binding of fructose 6-phosphate and inorganic phosphate, or Fru-2,6-P2, results in a decrease in the dissociation rate constant for AMP from fructose 1,6-bisphosphatase. These results are sufficient to explain the enhanced binding of AMP in the presence of Fru-2,6-P2 and, therefore, the synergistic inhibition of fructose 1,6-bisphosphatase observed with these two regulatory ligands;In order to understand the mechanism for AMP regulation of fructose 1,6-bisphosphatase and gluconeogenesis, kinetic studies of the enzyme with respect to Fru-1,6-P2 and Mg[superscript]2+ have been carried out. A rapid equilibrium random Bi Bi mechanism is suggested for the rabbit liver fructose 1,6-bisphosphatase from initial rate studies at pH 9.5. Our kinetic findings suggest that Mg[superscript]2+ and inhibitor AMP are mutually exclusive in their binding to fructose 1,6-bisphosphatase. The role of AMP as an inhibitor may be associated with its abililty to prevent enzyme-metal binding.