DNA interstrand cross-links (ICLs) are mainly repaired by the combined action of nucleotide excision repair and homologous recombination in E. coli. Genetic data also suggest the existence of a nucleotide excision repair-dependent, homologous recombination-independent ICL repair pathway. The involvement of translesion synthesis in this pathway has been postulated; however, the molecular mechanism of this pathway is not understood. To examine the role of translesion synthesis in ICL repair, we generated a defined substrate with a single psoralen ICL that mimics a postincision structure generated by nucleotide excision repair. We demonstrated that the Klenow fragment (DNA polymerase I) performs translesion synthesis on this model substrate. This in vitro translesion synthesis assay will help in understanding the basic mechanism of a postincision translesion synthesis process in ICL repair.