The effects of N2-isopropylGua and N6-isopropylAde adducts in template DNA on polymerization by the human replicative DNA polymerase alpha (B-family) and the translesion synthesis DNA polymerases eta and iota (Y-family) were investigated. A direct comparison between the accuracies of DNA synthesis using catalytic fragments of the human DNA polymerases eta and iota is reported. We show that the N2-isopropylGua adduct is a powerful block to polymerization by DNA polymerase alpha. In contrast, the DNA polymerases eta and iota synthesize DNA past the N2-isopropylGua lesion with efficiencies and accuracies opposite the lesion comparable to the unadducted Gua. All three DNA polymerases bypass the N6-isopropylAde adduct with only modest effects on efficiencies and accuracy. These results illustrate the dramatically different consequences to polymerization conferred by the position of the isopropyl adduct when catalyzed by DNA polymerase alpha and the lack of this effect on polymerization by the translesion synthesis DNA polymerases eta and iota. A steady-state kinetic analysis of nucleotide insertion opposite the N2-isopropylGua and the N6-isopropylAde by the DNA polymerases eta and iota was performed to measure the accuracy of DNA synthesis at these lesions. This analysis showed that the DNA polymerases eta and iota preferably insert the correct nucleotide Cyt opposite the N2-isopropylGua lesion and the correct nucleotide Thy opposite the N6-isopropylAde with levels of accuracy similar to those detected opposite the unadducted nucleotides, thus, demonstrating minimal blocking and mutagenic potential by these lesions to the translesion synthesis polymerases. Similarly, a kinetic analysis of polymerization opposite the N6-isopropylAde by the DNA polymerase alpha showed comparable levels of insertion accuracy relative to the unadducted Ade. These results suggest that positioning of the isopropyl adduct on the purine ring to locate this group into the minor groove of the DNA is an important determinant to effect blocked replication by a replicative (B-family) polymerase, but not to affect replication by a translesion synthesis (Y-family) polymerase.