Recent RNA polymerase (RNAP) structures led to a proposed three-step model of nucleoside triphosphate (NTP) binding, discrimination, and incorporation. NTPs are thought to enter through the secondary channel, bind to an E site, rotate into a pre-insertion (PS) site and ultimately align in the catalytic (A) site. We characterized the kinetics of correct and incorrect incorporation for several E. coli RNAPs with substitutions in the proposed NTP entry pore (secondary channel). Substitutions of the semi-conserved residue ßD675, which is >10Å away from these sites, significantly reduce fidelity; however, substitutions of the totally conserved residues ßR678 and ßD814 do not significantly alter the correct or incorrect incorporation kinetics, even though the corresponding residues in RNAPII crystal structures appear to be interacting with the NTP phosphate groups and coordinating the second magnesium ion in the active site, respectively. Structural analysis suggests that the lower fidelity of the ßD675 mutants most likely results from reduction of the negative potential of a small pore between the E and PS sites and elimination of several structural interactions around the pore. We suggest a mechanism of nucleotide discrimination that is governed both by rotation of the NTP through this pore and subsequent rearrangement or closure of RNAP to align the NTP in the A site.