In biological systems, enzymes often use metal ions, especially Mg 2+ , to catalyze phosphodiesterolysis, and model aqueous studies represent an important avenue of examining the contributions of these ions to catalysis. We have examined Mg 2+ and Ca 2+ catalyzed hydrolysis of the model phosphodiester thymidine-5′-p-nitrophenyl phosphate (T5PNP). At 25°C, we find that, despite their different Lewis acidities, these ions have similar catalytic ability with second-order rate constants for attack of T5PNP by hydroxide (k OH ) of 4.1×10 −4 M −1 s −1 and 3.7×10 −4 M −1 s −1 in the presence of 0.30M Mg 2+ and Ca 2+ , respectively, compared to 8.3×10 −7 M −1 s −1 in the absence of divalent metal ion. Examining the dependence of k OH on [M 2+ ] at 50°C indicates different kinetic mechanisms with Mg 2+ utilizing a single ion mechanism and Ca 2+ operating by parallel single and double ion mechanisms. Association of the metal ion(s) occurs prior to nucleophilic attack by hydroxide. Comparing the k OH values reveals a single Mg 2+ catalyzes the reaction by 1800-fold whereas a single Ca 2+ ion catalyzes the reaction by only 90-fold. The second Ca 2+ provides an additional 10-fold catalysis, significantly reducing the catalytic disparity between Mg 2+ and Ca 2+ .