Receptor-associated protein (RAP80), a nuclear protein containing two ubiquitin-interacting motifs (UIM), was recentlyfound to be associated with breast cancer-1 (BRCA1) and to translocate to ionizing radiation-induced foci (IRIF). In this study, we show that the BRCT mutant of BRCA1, R1699W, which is associated with increased risk of breast cancer, is unable to interact with RAP80. Previously, we showed that ataxia-telangiectasia mutated protein kinase (ATM) can phosphorylate RAP80 in vitro at Ser205, but whether this site is a target of ATM in whole cells was not established. To address this question, we generated an anti-RAP80Ser205P antibody that specifically recognizes RAP80 phosphorylated at Ser205. Our data show that RAP80 becomes phosphorylated at Ser205 in cells exposed to ionizing irradiation and that RAP80Ser205P translocates to IRIF. We show that this phosphorylation is mediated by ATM and does not require a functional BRCA1. The phosphorylation occurs within 5 minutes after irradiation, long before the translocation of RAP80 to IRIF. In addition, we show that UV irradiation induces translocation of RAP80 to DNA damage foci that colocalize with γ-H2AX. We further show that this translocation is also dependent on the UIMs of RAP80 and that the UV-induced phosphorylation of RAP80 at Ser 205 is mediated by ATM- and RAD3-related kinase, not ATM. These findings suggest that RAP80 has a more general role in different types of DNA damage responses. ©2008 American Association for Cancer Research.