ATR associates with the regulatory protein ATRIP that has been proposed to localize ATR to sites of DNA damage through an interaction with single-stranded DNA (ssDNA) coated with replication protein A (RPA). We tested this hypothesis and found that ATRIP is required for ATR accumulation at intranuclear foci induced by DNA damage. A domain at the N terminus of ATRIP is necessary and sufficient for interaction with RPA-ssDNA. Deletion of the ssDNA-RPA interaction domain of ATRIP greatly diminished accumulation of ATRIP into foci. However, the ATRIP-RPA-ssDNA interaction is not sufficient for ATRIP recognition of DNA damage. A splice variant of ATRIP that cannot bind to ATR revealed that ATR association is also essential for proper ATRIP localization. Furthermore, the ATRIP-RPA-ssDNA interaction is not absolutely essential for ATR activation because ATR phosphorylates Chk1 in cells expressing only a mutant of ATRIP that does not bind to RPA-ssDNA. These data suggest that binding to RPA-ssDNA is not the essential function of ATRIP in ATR-dependent checkpoint signaling and ATR has an important function in properly localizing the ATR-ATRIP complex.