A mechanism by which the Klenow fragment of DNA polymerase I monitors the geometry of the base pairs may involve hydrogen bonds between the polymerase and the minor groove of the nascent base pair. The involvement of the 3-position of guanine in the template strand was examined by synthesizing oligodeoxynucleotides containing guanine and 3-deazaguanine and comparing the steady-state kinetics of the incorporation of all four dNTPs. The Vmax/Km decreased a significant amount (170-fold) only when dCTP was the co-substrate suggesting that a hydrogen bond exists only when the correct base pair is being replicated. This approach was also used to examine how the Klenow fragment interacts with the 3-position of the mutagenic base O6-methylguanine (O6mG). The Vmax/Km for the incorporation of dTTP opposite O6-methyl-3-deazaguanine (O6m3DG) was 1700-fold less than opposite O6mG. In contrast, a small 6-fold increase in Vmax/Km occurred for the incorporation of dCTP opposite O6m3DG relative to O6mG. This result suggests that the hydrogen bond between the Klenow fragment and O6mG is more important in the incorporation of dTTP opposite O6mG and may contribute to the mutagenicity of O6mG.