The nucleotide excision repair (NER) pathway deals with a wide variety of lesions caused by UV-light, environmental mutagens and chemotherapeutic agents such as cisplatin. Deficiencies in NER are associated with the hereditary disorder xeroderma pigmentosum, which is characterized by a more than 1000-fold increased incidence in skin cancer. NER involves more than 30 proteins that recognize the lesion, excise a DNA fragment containing the damage and restore the integrity of the double stranded DNA. The structure-specific endonuclease ERCC1-XPF plays a key role in NER and its activity is also required for other repair pathways, in particular interstrand crosslink (ICL) repair and homologous recombination. In NER, XPA recruits ERCC1-XPF to NER complexes where it performs one of the two incisions necessary to excise the damaged DNA. Based on the structure of an XPA peptide bound to ERCC1, we identified ERCC1 and XPA residues that are involved in this interaction. Mutations of these residues led to reduced NER activity in vitro and in vivo. In addition, clonogenic survival assays showed that cells expressing one of those mutants, ERCC1-N110A/Y145A, were specifically sensitive to UV-light but not to ICL-forming agents. Conversely, mutations in a region of XPF specifically impaired the ability of the nuclease in the repair of ICLs. In conclusion, we generated the first mutants in ERCC1-XPF specifically deficient in NER or ICL repair by selectively targeting protein binding sites. This work demonstrates that the activity of ERCC1-XPF in various repair pathways is regulated by protein-protein interactions involving specific regions of the ERCC1 and XPF proteins.