Template primers containing propanodeoxyguanosine (PdG) in two different sequence contexts (C-PdG-C and T-PdG-T) were replicated by the Klenow fragment of DNA polymerase I. The presence of PdG in the template strand reduced the extent of in vitro DNA synthesis 10(3) - 10(4)-fold compared with unmodified template primers. Partial blockade was observed 1 base 3' to the adduct and opposite the adduct. Purines were preferentially incorporated opposite the adduct; the Vmax/Kmvalues for incorporation of dGMP were similar in both sequence contexts, whereas the Vmax/Km for dAMP incorporation increased 4.7-fold when the base pair 3' to PdG was changed from C:G to T:A. Oligonucleotides containing 1- and 2-base deletions were major products of replication in both sequence contexts. Full-length products were observed with templates containing T-PdG-T but not C-PdG-C. The major full-length product resulted from incorporation of dAMP residues opposite PdG. Kinetic analysis revealed that the major factor contributing to the selective incorporation of dAMP in full-length products was preferential extension of template primers containing PdG:dA termini rather than preferential incorporation of dAMP opposite PdG. The observation of PdG --> T mutations in the T-PdG-T context but not the C-PdG-C context during in vitro DNA replication parallels findings of in vivo experiments that base pair substitutions are induced by PdG in the former sequence context but not the latter.