The human Rad17–Rfc2-5 and Rad9–Rad1–Hus1 complexes play crucial roles in the activation of the ATR-mediated DNA damage and DNA replication stress response pathways. In response to DNA damage, Rad9 is recruited to chromatin in a Rad17-dependent manner in human cells. However, the DNA structures recognized by the Rad17–Rfc2-5 complex during the damage response have not been defined. Here, we show that replication protein A (RPA) stimulates the binding of the Rad17–Rfc2-5 complex to single-stranded DNA (ssDNA), primed ssDNA, and a gapped DNA structure. Furthermore, RPA facilitates the recruitment of the Rad9–Rad1–Hus1 complex by the Rad17–Rfc2-5 complex to primed and gapped DNA structures in vitro. These findings suggest that RPA-coated ssDNA is an important part of the structures recognized by the Rad17–Rfc2-5 complex. Unlike replication factor C (RFC), which uses the 3′ primer/template junction to recruit proliferating cell nuclear antigen (PCNA), the Rad17–Rfc2-5 complex can use both the 5′ and the 3′ primer/template junctions to recruit the Rad9–Rad1–Hus1 complex, and it shows a preference for gapped DNA structures. These results explain how the Rad17–Rfc2-5 complex senses DNA damage and DNA replication stress to initiate checkpoint signaling.