Enzymes which cleave the phosphodiester bonds of DNA and RNA often employ divalent metal ions such as Mg²⁺. One important role of these metal ions is the activation of a solvent nucleophile. To examine nucleophilic activation by Mg²⁺ in the absence of an enzyme active site, the rate and temperature dependence of the cleavage of the model phosphodiester thymidine-5'-p-nitrophenyl phosphate (T5PNP) by HO^- were analyzed in the absence and presence of 0.3 M Mg²⁺. In the presence of 0.3 M Mg²⁺, the second order rate constant (k) increased over 200 fold, from 7.1 x 10^-7 M^-1s^-1 to 1.6 x 10^-4M^-1s^-1. The nucleophilic species considered for the Mg²⁺ catalyzed reaction included HO^-, [Mg(H₂O)₆OH]⁺ free in solution, and [Mg(H₂O)₆OH]⁺ acting as a complex with T5PNP. The model most consistent with the data is [Mg(H₂O)₆OH]⁺ acting as a complex with a k of 6.2 x 10^-6 M^-1s^-1. Temperature dependence of the reactions indicate that in the absence of Mg²⁺, DH‡ is 76 (±2)kJ/mol and DS‡ is ^-106 (±8)J/mol K. In the presence Mg²⁺, DH‡ is 95 (±7) kJ/mol and DS‡ is ^-24 (±8) J/mol K. When the entropic advantage of the lower pK_a of metal coordinated water was accounted for by using [Mg(H₂O)₆OH]⁺ as the active nucleophile, the DS‡ became ^-63 (±8) J/mol K. These values suggest a substantial difference in the solvation changes upon going to the transition state for the Mg²⁺ catalyzed reaction and/or an entropic mode of catalysis for nucleophilic activation Mg²⁺.