We have analyzed the divalent cation specificity of poliovirus RNA-dependent RNA polymerase, 3D(pol). The following preference was observed: Mn(2+) > Co(2+) > Ni(2+) > Fe(2+) > Mg(2+) > Ca(2+) > Cu(2+), and Zn(2+) was incapable of supporting 3D(pol)-catalyzed nucleotide incorporation. In the presence of Mn(2+), 3D(pol) activity was increased by greater than 10-fold relative to that in the presence of Mg(2+). Steady-state kinetic analysis revealed that the increased activity observed in the presence of Mn(2+) was due, primarily, to a reduction in the K(M) value for 3D(pol) binding to primer/template, without any significant effect on the K(M) value for nucleotide. The ability of 3D(pol) to catalyze RNA synthesis de novo was also stimulated approximately 10-fold by using Mn(2+), and the enzyme was now capable of also utilizing a DNA template for primer-independent RNA synthesis. Interestingly, the use of Mn(2+) as divalent cation permitted 3D(pol) activity to be monitored by following extension of 5'-(32)P-end-labeled, heteropolymeric RNA primer/templates. The kinetics of primer extension were biphasic because of the enzyme binding to primer/template in both possible orientations. When bound in the incorrect orientation, 3D(pol) was capable of efficient addition of nucleotides to the blunt-ended duplex; this activity was also apparent in the presence of Mg(2+). In the presence of Mn(2+), 3D(pol) efficiently utilized dNTPs, ddNTPs, and incorrect NTPs. On average, three incorrect nucleotides could be incorporated by 3D(pol). The ability of 3D(pol) to incorporate the correct dNTP, but not the correct ddNTP, was also observed in the presence of Mg(2+). Taken together, these results provide the first glimpse into the nucleotide specificity and fidelity of the poliovirus polymerase and suggest novel alternatives for the design of primer/templates to study the mechanism of 3D(pol)-catalyzed nucleotide incorporation.