Fructose 1, 6-diphosphatase [EC 3.1 3.11] from rabbit liver was inactivated by incubation with oxidized gluthathione. Other disulfide compouds tested, or reduced gluthathione, were without effects. The inactivation reaction proceeded in the neutral pH range, and two apparent pK values were obtained at 4.6 and 7.6. The inactivation was completely prevented by metal cofactors of the enzyme, Mg2+ and Mn2+ although restoration by them of the activity of oxidized gluthathione-inactivated enzyme was relatively slow. The inactivation was also prevented and completely reversed by pyridoxal 5′-phosphate, neucleotide tri- and diphosphates or other chelating compounds such as citrate, malonate, EDTA, or 8-hydroxyquinoline. sulfhydryl compounds such as dithiotheritol or reduced gulthathione had no protective or reversal effects on the inactivation reaction, and no decrease of enxyme sulfhydryl groups was detected after inactivation by oxidized gulthathione. These results suggested that the inactivation of fructose 1,6-diphosphatase by oxidized gultatione may be related to the divalent cation binding site, not to the sulfhydryl groups of the enzyme.