The heterotrimeric checkpoint clamp comprises the Saccharomyces cerevisiae Rad17, Mec3, and Ddc1 subunits (Rad17/3/1, the 9-1-1 complex in humans). This DNA damage response factor is loaded onto DNA by the Rad24-RFC (replication factor C-like complex with Rad24) clamp loader and ATP. Although Rad24-RFC alone does not bind to naked partial doublestranded DNA, coating of the single strand with single-stranded DNA-binding protein RPA (replication protein A) causes binding of Rad24-RFC via interactions with RPA. However, RPAmediated binding is abrogated when the DNA is coated with RPA containing a rpa1-K45E (rfa1-t11) mutation. These properties allowed us to determine the role of RPA in clamp-loading specificity. The Rad17/3/1 clamp is loaded with comparable efficiency onto naked primer/template DNA with either a 3′-junction or a 5′-junction. Remarkably, when the DNA was coated with RPA, loading of Rad17/3/1 at 3′-junctions was completely inhibited, thereby providing specificity to loading at 5′-junctions. However, Rad17/3/1 loaded at 5′-junctions can slide across double-stranded DNA to nearby 3′-junctions and thereby affect the activity of proteins that act at 3′-termini. These studies show a unique specificity of the checkpoint loader for 5′-junctions of RPA-coated DNA. The implications of this specificity for checkpoint function are discussed.