Isotope-trapping experiments with mental-free rabbit liver fructose 1,6-bisphosphatase have shown that enzyme-bound D-fructose 1,6-bisphosphate completely dissociates prior to enzyme turnover initiated by Mn2+ as the catalytic metal. The exchange rate of the binary enzyme-D-fructose 1,6-bisphosphate complex with the substrate pool is, therefore, more rapid than its conversion to products, suggesting that structural Mn2+ is necessary for productive substarate binding. Rapid-quench isotope-trapping experiments confirm the requirement for structural Mn2+ ions for productive binding to occur. These experiments also show that an ordered formation of the enzyme-Mn2+ s-D-fructose 1,6-bisphosphate ternary complex which features metal-ion addition prior to substrate constitutes a catalytically competent pathway in the mechanism of fructose 1,6-bisphosphatase and that all four subunits are active in a single turnover event.