A variety of DNA polymerases, synthesizing in vitro on an UV-irradiated phi X174 DNA template, terminate synthesis one nucleotide before the 3' pyrimidines of putative dimers on the template. We have devised a system using Escherichia coli DNA polymerase I (Klenow fragment) that can synthesize past at least some of these dimers. The bypass is carried out in a multistep process--first, the incorporation of nucleotides opposite the pyrimidines in the dimer and, then, the addition of nucleotides complementary to the bases distal to the dimer. The insertion of a nucleotide opposite the first (3') pyrimidine of a putative dimer in the presence of Mn2+ occurs in a concentration-dependent fashion with a 3- to 4-fold preference for purine nucleotides over pyrimidine nucleotides. In the presence of Mg2+, insertion is less frequent. Correlation of these results with in vivo mutation data suggests a role for the polymerase in determining the spectrum of base substitution mutagenesis in SOS induced cells.