Fructose-1,6-bisphosphatase requires a divalent metal cation for catalysis, Mg²⁺being its most studied activator. Phosphatase activity increases sigmoidally with the concentration of Mg²⁺, but the mechanistic basis for such cooperativity is unknown. Bound magnesium cations can interact within a single subunit or between different subunits of the enzyme tetramer. Mutations of Asp¹¹⁸, Asp¹²¹, or Glu⁹⁷ to alanine inactivate the recombinant porcine enzyme. These residues bind directly to magnesium cations at the active site. Three different hybrid tetramers of fructose-1,6-bisphosphatase, composed of one wild-type subunit and three subunits bearing one of the mutations above, exhibit kinetic parameters (K_m for fructose-1,6-bisphosphate, 1.1–1.8 μM; K_a for Mg²⁺, 0.34–0.76 mM; K_i for fructose-2,6-bisphosphate, 0.11–0.61 μM; and IC₅₀ for AMP, 3.8–7.4 μM) nearly identical to those of the wild-type enzyme. Notwithstanding these similarities, thek_cat parameter for each hybrid tetramer is approximately one-fourth of that for the wild-type enzyme. Evidently, each subunit in the wild-type tetramer can independently achieve maximum velocity when activated by Mg²⁺. Moreover, the activities of the three hybrid tetramers vary sigmoidally with the concentration of Mg²⁺ (Hill coefficients of ∼2). The findings above are fully consistent with a mechanism of cooperativity that arises from within a single subunit of fructose-1,6-bisphosphatase.