The role of divalent cations in the mechanism of pyrophosphate-activated, template-directed oligoribonucleotide ligation has been investigated. The dependence of the reaction rate on Mg2+ concentration suggests a kinetic scheme in which a Mg2+ ion must bind before ligation can proceed. Mn2+, Ca2+, Sr2+, and Ba2+ can also catalyze the reaction. Although Pb2+ and Zn2+ do not catalyze the reaction in the absence of other divalent ions, they significantly modulate the reaction rate when added in the presence of Mg2+, With Pb2+ stimulating the reaction (up to 65-fold) and Zn2+ inhibiting the reaction. The logarithm of the ligation rate increases linearly, with slope of 0.95, as a function of pH, indicating that the reaction involves a single critical deprotonation step. The ligation rates observed with the different divalent metal ion catalysts (Mn2+ > Mg2+ > Ca2+ > Sr2+ = Ba2+) vary inversely with the pK(a) values of their bound water molecules. The pH profile and these relative ligation rates suggest a mechanism in which a metal-bound hydroxide ion located near the ligation junction promotes catalysis, most likely by deprotonation of the hydroxyl nucleophile. The effects of changing either the leaving group or the attacking hydroxyl, together with the large Delta S-double dagger value for oligonucleotide ligation (about -20 eu), are consistent with an associative transition state.