We have performed a series of first-principles electronic structure calculations to study competing reaction pathways and the corresponding free-energy barriers for the ester hydrolysis of intracellular second-messenger adenosine 3',5'-cyclic monophosphate (cAMP) and related phosphodiesters including trimethylene phosphate (TMP). Reaction coordinate calculations show three fundamental reaction pathways for the ester hydrolysis, including (A) attack of a hydroxide ion at the P atom of the phosphate anion (an S(N)2 process without a pentacoordinated phosphorus intermediate), (B) direct attack of a water molecule at the P atom of the anion (a three-step process), and (C) direct attack of a water molecule at the P atom of the neutral ester molecule (a two-step process). The calculated energy results show that for the reactions in the gas phase the free-energy barrier for pathway A is the highest and the barrier for the rate-controlling step of pathway C is the lowest. However, for the reactions in aqueous solution, the free-energy barrier calculated for pathway A becomes the lowest, and the two main hydrolysis pathways are A and B. We also have demonstrated how the pK(a) of the ester and the pH of the reaction solution affect the relative contributions of different hydrolysis pathways to the total hydrolysis rate. Reaction pathway A should be dominant for the cAMP hydrolysis in neutral aqueous solution. However, the relative contribution of pathway A to the total hydrolysis rate should decrease with decreasing pH of the solution. For pH < similar to3.7, the contribution of pathway B is larger. For pH > similar to3.7, the contribution of pathway A is larger. The reliability of our theoretical predictions is supported by the excellent agreement of the calculated free-energy barrier with available experimental data for the hydrolysis of TMP in solution.