Local conformational changes in primer-template (P/T) DNA are involved in the selective incorporation of dNTP by DNA polymerases (DNAP). Here we use near UV CD and fluorescence spectra of pairs of base analogue probes, substituted either at the primer terminus or in the coding region of the template strand, to monitor and interpret conformational changes at and near the coding base of the template in P/T DNA complexes with Klenow fragment (KF) DNAP as the polymerase moves through the nucleotide addition cycle. Incoming dNTPs and rNTPs encounter binary complexes in which the 3'-end of the primer shuttles between the polymerization (pol) and exonuclease (exo) sites of DNAPs, even for perfectly complementary P/T DNA sequences. We have used spectral changes of probes inserted in both strands to monitor this two-state distribution and determine how it depends on the formation of ternary complexes with both complementary ("correct") and noncomplementary ("incorrect") NTPs and on the local sequence of the P/T DNA. The results show that the relative occupancy of the exo and pol sites is coupled to conformational changes in the P/T DNA of the complex that are partially regulated by the incoming NTP. We find that the coding base on the template strand is unperturbed by the binding of incorrect dNTPs, while binding of complementary rNTPs induces a novel template conformation. We conclude that, in addition to its editing function, primer strand occupancy of the 3'-exo site may also serve as a regulatory checkpoint for accurate dNTP selection in DNA synthesis.