2'-Chloro-2'-deoxyadenosine triphosphate (cladribine), a purine nucleotide analog and potent antileukemic agent, was enzymatically incorporated into 98-base oligomers in place of dATP to investigate the molecular consequences of 2-chloroadenine (CIAde) in DNA. We have used the resultant oligomers as templates for purified DNA polymerases, to compare the rate and extent of in vitro DNA synthesis; the sites of polymerase pausing, if any; and the effects of increasing deoxyribonucleoside triphosphate (dNTP) concentrations on synthetic reactions. Compared with control template, CIAde-containing DNA strikingly reduced the overall amount and rate of chain elongation by human polymerase beta and Klenow fragment. Distinct pause sites, which were polymerase dependent, occurred primarily one or two bases before or just after nucleotide incorporation opposite template CIAde. Human polymerase alpha and phage T4 DNA polymerase likewise exhibited reduced synthesis on CIAde-substituted templates. Bypassing of CIAde residues was possible only at higher dNTP concentrations, with approximately 20- and 50-fold greater dNTP concentrations being required for synthesis beyond CIAde sites, compared with adenine residues, by polymerase alpha and beta, respectively. These results suggest that CiAde residues located within cellular template DNA may inhibit daughter strand synthesis and thus contribute to the cytotoxic effects of the drug.