During each catalytic cycle, DNA polymerases select deoxyribonucleoside triphosphate (dNTP) substrates complementary to a templating base with high fidelity from a pool that includes noncomplementary dNTPs and both complementary and noncomplementary ribonucleoside triphosphates (rNTPs). The Klenow fragment of Escherichia coli DNA polymerase I (KF) achieves this through a series of conformational transitions that precede the chemical step of phosphodiester bond formation. Kinetic evidence from fluorescence and FRET experiments indicates that discrimination of the base and sugar moieties of the incoming nucleotide occurs in distinct, sequential steps during the selection pathway. Here we show that KF-DNA complexes formed with complementary rNTPs or with noncomplementary nucleotides can be distinguished on the basis of their properties when captured in an electric field atop the α-hemolysin nanopore. The average nanopore dwell time of KF-DNA complexes increased as a function of complementary rNTP concentration. The increase was less than that promoted by complementary dNTP, indicating that the rNTP complexes are more stable than KF-DNA binary complexes but less stable than KF-DNA-dNTP ternary complexes. KF-DNA-rNTP complexes could also be distinguished from KF-DNA-dNTP complexes on the basis of ionic current amplitude. In contrast to complementary rNTPs, noncomplementary dNTPs and rNTPs diminished the average nanopore dwell time of KF-DNA complexes in a concentration-dependent manner, suggesting that binding of a noncomplementary nucleotide keeps the KF-DNA complex in a less stable state. These results imply that nucleotide selection proceeds through a series of complexes of increasing stability in which substrates with the correct moiety promote the forward transitions.