The chemical reactivity of ribonucleotide analog 2’-deoxy-2’-thiouridine 3’-(p-nitrophenyl phosphate) (1), in which the 2’ hydroxyl is replaced with a 2’-thiol group, has been characterized. The major reaction pathway for 1, as monitored by P-31 NMR spectroscopy, is transphosphorylation to afford 2’,3’-cyclic phosphorothioate 3, followed by hydrolysis of 3 to produce 2’-deoxy-2’-thiouridine 2’-phosphorothioate (4). Thus, the reaction pathway of 1 is similar to that of the hydrolysis of ribonucleotides, yet there are significant differences. The pH-rate profile for transphosphorylation of 1 was determined by monitoring the formation of p-nitrophenol or p-nitrophenolate by UV-visible spectroscopy. Analysis of the profile reveals the attacking nucleophile to be thiolate, and the pK(a) of the 2’-thiol was determined to be 8.3 +/- 0.1. At pH 7.4, the thiol-containing ribonucleotide analog 1 is hydrolyzed at an observed rate 27-fold slower than its 2’-hydroxyl counterpart. These results indicate that the rate of thiolate attack on the adjacent phosphodiester bond is 10(7)-fold slower than that of the corresponding alkoxide. Thiolate nucleophiles, therefore, are remarkably reticent toward attack at electrophilic phosphate centers. In addition to providing new information about the reactivity of phosphodiester bonds, our studies highlight the potential of 2’-thiol-containing nucleotides for the study of an array of RNA processes, especially those in which the 2’-substituent plays a critical role.