Phosphodiester linkages, including those that join the nucleotides of DNA, are highly resistant to spontaneous hydrolysis. The rate of water attack at the phosphorus atom of phosphodiesters is known only as an upper limit, based on the hydrolysis of the dimethyl phosphate anion. That reaction was found to proceed at least 99% by C-O cleavage, at a rate suggesting an upper limit of 10(-15) s(-1) for P-O cleavage of phosphodiester anions at 25 degrees C. To evaluate the rate enhancement produced by P-O cleaving phosphodiesterases such as staphylococcal nuclease, we decided to establish the actual value of the rate constant for P-O cleavage of a simple phosphodiester anion. In dineopentyl phosphate, C-O cleavage is sterically precluded so that hydrolysis occurs only by P-O cleavage. Measurements at elevated temperatures indicate that the dineopentyl phosphate anion undergoes hydrolysis in water with a t(1/2) of 30,000,000 years at 25 degrees C, furnishing an indication of the resistance of the internucleotide linkages of DNA to water attack at phosphorus. These results imply that staphylococcal nuclease (k(cat) = 95 s(-1)) enhances the rate of phosphodiester hydrolysis by a factor of approximately 10(17). In alkaline solution, thymidylyl-3'-5'-thymidine (TpT) has been reported to decompose 10(5)-fold more rapidly than does dineopentyl phosphate. We find however that TpT and thymidine decompose at similar rates and with similar activation parameters, to a similar set of products, at pH 7 and in 1 M KOH. We infer that the decomposition of TpT is initiated by the breakdown of thymidine, not by phosphodiester hydrolysis.