The endonuclease ERCC1-XPF incises the damaged strand of DNA 5′ to a lesion during nucleotide excision repair (NER) and has additional, poorly characterized functions in interstrand cross-link repair, double-strand break repair, and homologous recombination. XPA, another key factor in NER, interacts with ERCC1 and recruits it to sites of damage. We identified ERCC1 residues that are critical for the interaction with XPA and assessed their importance for NER in vitro and in vivo. Mutation of two conserved residues (Asn-110 and Tyr-145) located in the XPA-binding site of ERCC1 dramatically affected NER but not nuclease activity on model DNA substrates. In ERCC1-deficient cells expressing ERCC1N110A/Y145A, the nuclease was not recruited to sites of UV damage. The repair of UV-induced (6-4)photoproducts was severely impaired in these cells, and they were hypersensitive to UV irradiation. Remarkably, the ERCC1N110A/Y145A protein rescues the sensitivity of ERCC1-deficient cells to cross-linking agents. Our studies suggest that ERCC1-XPF engages in different repair pathways through specific protein-protein interactions and that these functions can be separated through the selective disruption of these interactions. Wediscuss the impact of these findings for understanding how ERCC1 contributes to resistance of tumor cells to therapeutic agents such as cisplatin.