Chemotherapeutic agents such as mitomycin C or nitrogen mustards induce DNA inter-strand cross-links (ICL) and are highly toxic, thus constituting an useful tool to treat some human degenerative diseases, such as cancer. Additionally, psoralens plus UV-A (PUVA), which also induce ICL, find use in treatment of patients afflicted with psoriasis and vitiligo. The repair of DNA ICL generated by different molecules involves a number of multi-step DNA repair pathways. In bacteria, as in eukaryotic cells, if DNA ICL are not tolerated or repaired via nucleotide excision repair (NER), homologous recombination or translesion synthesis pathways, these DNA lesions may lead to mutations and cell death. Herein, we bring new insights to the role of Escherichia coli nucleotide excision repair genes uvrA, uvrB and uvrC in the repair of DNA damage induced by some chemotherapeutic agents and psoralen derivatives plus UV-A. These new observations point to a novel role for the UvrB protein, independent of its previously described role in the Uvr(A)BC complex, which could be specific for repair of monoadducts, intra-strand biadducts and/or ICL.