In order to examine the effect of purine adducts of the plant carcinogen aristolochic acid (AA) on DNA replication four 30-mer templates were prepared which contained single site-specific AA lesions. The oligonucleotides were isolated by HPLC and shown to contain the two known aristolochic acid I-DNA adducts (dA-AAI, dG-AAI) or the two known aristolochic acid II-DNA adducts (dA-AAII, dG-AAII) at position 27 from the 3' end by 32P-postlabeling. These adducts templates were replicated in primer (23-mer) extension reactions catalysed by human DNA polymerase alpha. Both AAI-DNA adducts (dA-AAI, dG-AAI) blocked DNA synthesis predominantly (80-95%) at the nucleotide 3' to the adduct, although primer extension to the full length of the template was found with unmodified control templates. Increasing dNTP concentrations had only a small effect on the DNA synthesis and translesional synthesis was negligible. In contrast, both AAII-DNA adducts showed marked differences in primer extension reactions. Blocking of DNA synthesis by the dA-AAII adduct was strongly dNTP dependent. With increasing dNTP concentrations 27 and 28 nucleotide products, indicating termination of DNA synthesis after incorporation of a nucleotide opposite this adduct and incorporation of an additional nucleotide accumulated. Only the dG-AAII adducted template allowed substantial translesional synthesis to the full length of the template (up to 25%). When a 26-mer primer was used to examine nucleotide incorporation directly across from the four purine adducts, we found no detectable incorporation of nucleotides for the dA-AAI adduct, whereas the dG-AAI adduct and both AAII-adducts (dA-AAII and dG-AAII) allowed preferential incorporation of the correct nucleotide. These results indicate that for human polymerase alpha three AA purine adducts (dA-AAI, dG-AAI and dA-AAII) provide severe blocks to DNA replication and that dG-AAII, which allows translesional synthesis, may not be a very efficient mutagenic lesion.