In this study, we examine the potential role of receptor-associated protein 80 (RAP80), a nuclear protein containing two ubiquitin-interacting motifs (UIM), in DNA damage response and double-strand break (DSB) repair. We show that following ionizing radiation and treatment with DNA-damaging agents, RAP80 translocates to discrete nuclear foci that colocalize with those of γ-H2AX. The UIMs and the region of amino acids 204 to 304 are critical for the relocalization of RAP80 to ionizing radiation-induced foci (IRIF). These observations suggest that RAP80 becomes part of a DNA repair complex at the sites of IRIF. We also show that RAP80 forms a complex with the tumor repressor BRCA1 and that this interaction is mediated through the BRCA1 COOH-terminal repeats of BRCA1. The UIMs are not required for the interaction of RAP80 with BRCA1. Knockdown of RAP80 in HEK293 cells significantly reduced DSB-induced homology-directed recombination (HDR). Moreover, inhibition of RAP80 expression by small interfering RNA increased radiosensitivity, whereas increased radioresistance was observed in human breast cancer MCF-7 cells with overexpression of RAP80. Taken together, our data suggest that RAP80 plays an important role in DNA damage response signaling and HDR-mediated DSB repair. We further show that RAP80 can function as a substrate of the ataxia-telangiectasia mutated protein kinase in vitro, which phosphorylates RAP80 at Ser 205 and Ser402. We show that this phosphorylation is not required for the migration of RAP80 to IRIF. ©2007 American Association for Cancer Research.