In order to assess the contribution to discrimination afforded by base pair hydrogen bonding during DNA replication by the human mitochondrial DNA polymerase, we examined nucleoside mimics lacking hydrogen bond forming capability, but retaining the overall steric shape of the natural nucleotide. We employed oligonucleotide templates containing either a deoxyadenosine shape mimic (dQ) or a deoxythymidine shape mimic (dF). Additionally, the nucleoside triphosphate analogs dFTP, dQTP, and dZTP (another dATP shape mimic) were assayed. We used presteady state methods to determine the kinetic parameters governing nucleotide incorporation, k_pol and K_d. In general, the loss of hydrogen bonding potential led to 2 3 kcal/mol reduction in ground state binding free energy, while effects on the maximum rate of polymerization were quite variable, ranging from negligible (dATP:dF) to nearly 4 kcal/mol (dZTP:dT). Although we observed only a 46-fold reduction in discrimination when dF was present in the template, there was a complete elimination of discrimination when dQ was present in the template. Our data with dF indicate that hydrogen bonding contributes 2.2 kcal/mol toward the efficiency of incorporation, while data with dQ (which may overestimate the effect due to poor steric mimicry) suggest a contribution of up to 6.8 kcal/mol. Taken together, the data suggest that sterics alone are not sufficient to achieve optimal efficiency and fidelity for Pol and that base pair hydrogen bonding