The human repair proteins XPA and ERCC1 have been shown to be absolutely required for the incision step of nucleotide excision repair, and recently we identified an interaction between these two proteins both in vivo and in vitro (L. Li, SJ Elledge, CA Peterson, ES Bales, and RJ Legerski, Proc. Natl. Acad. Sci. USA 91: 5012–5016, 1994). In this report, we demonstrate the functional relevance of this interaction. The ERCC1-binding domain on XPA was previously mapped to a region containing two highly conserved XPA sequences, Gly-72 to Phe-75 and Glu-78 to Glu-84, which are termed the G and E motifs, respectively. Site-specific mutagenesis was used to independently delete these motifs and create two XPA mutants referred to as DG and DE. In vitro, the binding of ERCC1 to DE was reduced by approximately 70%, and binding to DG was undetectable; furthermore, both mutants failed to complement XPA cell extracts in an in vitro DNA repair synthesis assay. In vivo, the DE mutant exhibited an intermediate level of complementation of XPA cells and the DG mutant exhibited little or no complementation. In addition, the DG mutant inhibited repair synthesis in wild-type cell extracts, indicating that it is a dominant negative mutant. The DE and DG mutations, however, did not affect preferential binding of XPA to damaged DNA. These results suggest that the association between XPA and ERCC1 is a required step in the nucleotide excision repair pathway and that the probable role of the interaction is to recruit the ERCC1 incision complex to the damaged site. Finally, the affinity of the XPA-ERCC1 complex was found to increase as a function of salt concentration, indicating a hydrophobic interaction; the half-life of the complex was determined to be approximately 90 min.