Several analogues of diuridine phosphate (UpU) were synthesized in order to investigate why replacing the 2‘-hydroxyl with a 2‘-amino group prevents hydrolysis. These analogues were designed to investigate what influence the 2‘-substituent and 5‘-leaving group have upon the rate of hydrolysis. All the analogues were considerably more labile than UpU toward acid−base-catalyzed hydrolysis. In the pH region from 6 to 9, the rate of hydrolysis of uridylyl (3‘−5‘) 5‘-thio-5‘-deoxyuridine (UpsU) hydrolysis rose, in a log linear fashion, from a value of 5 × 10^-6 s^-1 at pH 6 to 3200 × 10^-6 s^-1 at pH 9, indicating that attack on the phosphorus by the 2‘-oxo anion is rate-limiting in the hydrolysis mechanism. In contrast, the rate of uridylyl (3‘−5‘) 5‘-amino-5‘-deoxyuridine (UpnU) hydrolysis fell from a value of 1802 × 10^-6 s^-1 at pH 5 to 140 × 10^-6 s^-1 at pH 7.5, where it remained constant up to pH 11.5, thus indicating an acid-catalyzed reaction. The analogue 2‘-amino-2‘-deoxyuridylyl (3‘−5‘) 5‘-thio-5‘-deoxyuridine (amUpsU) was readily hydrolyzed above pH 7, in contrast to the hydrolytic stability of amUpT, with rates between 85 × 10^-6 s^-1 and 138 × 10^-6 s^-1. The hydrolysis of 2‘-amino-2‘-deoxyuridylyl (3‘−5‘) 5‘-amino-5‘-deoxythymidine (amUpnT) rose from 17 × 10^-6 s^-1 at pH 11.5 to 11 685 × 10^-6 s^-1 at pH 7.0, indicating an acid-catalyzed reaction, where protonation of the 5‘-amine is rate limiting. The cleavage rates of UpsU, UpnU, and amUpsU were accelerated in the presence of Mg²⁺, Zn²⁺, and Cd²⁺ ions, but a correlation with interaction between metal ion and leaving group could only be demonstrated for amUpsU. UpsU and UpnU are also substrates for RNase A with UpsU having similar Michaelis−Menten parameters to UpU. In contrast, UpnU is more rapidly degraded with an approximate 35-fold increase in catalytic efficiency, which is reflected purely in an increase in the value of k_cat.