Biological phosphates form the anionic backbone linkage in DNA and RNA and play a central role in the regulation of cellular processes including signaling, respiration, replication, and translation. Consequently, the study of the chemistry of biological phosphates is an area of great importance. Of particular interest are the mechanisms by which RNA can catalyze fairly complicated reactions such as the transphosphorylation and hydrolysis of phosphodiester bonds. A useful experimental strategy to probe the catalytic mechanisms of RNA enzymes is the study of thio effects: changes in the reaction rate that occur upon substitution of key phosphate oxygen atoms with sulfur atoms. Kinetic analysis of thio effects provides insight into the specific role that these oxygen positions play in catalysis. Theoretical methods are powerful tools to aid the interpretation of kinetic data through characterization of the structure and energetics of transition states and intermediates along competing reaction paths. The dominant reaction path for transphosphorylation in RNA (Scheme 1) proceeds via an in-line attack of an activated 2’-hydroxy group of the RNA sugar ring on the reactive phosphate group to produce a pentavalent phosphorane transition state or intermediate, followed by the cleavage of the P-O bond to produce a 2’,3’-cyclic phosphate. Experimental and theoretical data suggest a dianionic oxyphosphorane transphosphorylation intermediate is kinetically indistinguishable from a transition state and is too short-lived to undergo other processes such as protonation or pseudorotation. As the pH is lowered, acid-catalyzed migration products begin to emerge, which result from pseudorotation of a singly or doubly protonated phosphorane intermediate (Scheme 2). The ratio of products resulting from phosphate hydrolysis/ transphosphorylation and isomerization (migration) and their pH dependence involve a balance between the endoand exo-