The formation of pyrophosphate as a result of nucleophilic attack by orthophosphate at the acylphosphate bond of acetyl phosphate was detectable in completely aqueous media, and was enhanced by dimethyl sulfoxide. The reaction had an absolute requirement for divalent cations, the rate constant of phosphorolysis being dependent on the species and concentration of cations as well as on temperature and pH. The amount of pyrophosphate formed depended on both the acetyl phosphate and orthophosphate concentrations. In purely aqueous media, phosphorolysis was barely detectable in the presence of Mg2+, and its rate increased 40-fold when Mg2+ was replaced by Ca2+ or Sr2+. In the presence of Mg2+ the rate of phosphorolysis increased 400-fold when 50 to 80% of the water was replaced by dimethyl sulfoxide. In the latter case, the rate also increased as the pH was raised from 4.0 to 9.0. The entropy of activation was large and negative in the presence of Mg2+ or Ca2+, indicating that the nucleophile is involved in the rate-limiting step of the reaction. Since this thermodynamic parameter became large and positive in the presence of Ca2+ when dimethyl sulfoxide was omitted, it is inferred that the transition state of the same reaction may be changed by the solvent composition and the solvation of reactants.