We have measured the accuracy of in vitro synthesis by DNA polymerase I (large fragment) during translesion synthesis past an aminofluorene (AF) adduct. These studies were carried out using a site-specifically modified template which contained a single AF adduct. The template was prepared by first modifying the lone guanine in a 17 base long oligonucleotide and extensively purifying and characterizing this product. The modified 17mer was then ligated to a synthetic duplex to produce a 31 nucleotide long template strand containing the AF adduct annealed to a 14mer, such that the 3'-hydroxyl primer terminus was four nucleotides before the modified guanine. Synthesis on this template by DNA polymerase I efficiently bypassed the AF adduct and produced full-length duplex 31mers. T7 DNA polymerase, on the other hand, was unable to utilize the AF-modified template though it was active on an identical unmodified one. The strand synthesized by DNA polymerase I was then separated from the modified strand, annealed to a complementary oligonucleotide, and the resulting heteroduplex cloned into M13. Each of the 49 clones isolated had sequences which indicated that cytidine had been incorporated opposite the AF-modified guanine.