The use of Mn(2+) as the divalent cation cofactor in polymerase-catalyzed reactions instead of Mg(2+) often diminishes the stringency of substrate selection and incorporation fidelity. We have solved the complete kinetic mechanism for single nucleotide incorporation catalyzed by the RNA-dependent RNA polymerase from poliovirus (3D(pol)) in the presence of Mn(2+). The steps employed during a single cycle of nucleotide incorporation are identical to those employed in the presence of Mg(2+) and include a conformational-change step after nucleotide binding to achieve catalytic competence of the polymerase-primer/template-nucleotide complex. In the presence of Mn(2+), the conformational-change step is the primary determinant of enzyme specificity, phosphoryl transfer appears as the sole rate-limiting step for nucleotide incorporation, and the rate of phosphoryl transfer is the same for all nucleotides: correct and incorrect. Because phosphoryl transfer is the rate-limiting step in the presence of Mn(2+), it was possible to determine that the maximal phosphorothioate effect in this system is in the range of 8-11. This information permitted further interrogation of the nucleotide-selection process in the presence of Mg(2+), highlighting the capacity of this cation to permit the enzyme to use the phosphoryl-transfer step for nucleotide selection. The inability of Mn(2+) to support a reduction in the efficiency of phosphoryl transfer when incorrect substrates are employed is the primary explanation for the loss of fidelity observed in the presence of this cofactor. We propose that the conformational change involves reorientation of the triphosphate moiety of the bound nucleotide into a conformation that permits binding of the second metal ion required for catalysis. In the presence of Mg(2+), this conformation requires interactions with the enzyme that permit a reduction in catalytic efficiency to occur during an attempt to incorporate an incorrect nucleotide. Adventitious interactions in the cofactor-binding site with bound Mn(2+) may diminish fidelity by compensating for interaction losses used to modulate catalytic efficiency when incorrect nucleotides are bound in the presence of Mg(2+).