Replication protein A (RPA) is a highly conserved single-stranded DNA-binding protein involved in DNA replication, recombination and repair. We show here that RPA is present at the telomeres of the budding yeast Saccharomyces cerevisiae, with a maximal association in S phase. A truncation of the N-terminal region of Rfa2p (associated with the rfa2Delta40 mutated allele) results in severe telomere shortening caused by a defect in the in vivo regulation of telomerase activity. Cells carrying rfa2Delta40 show impaired binding of the protein Est1p, which is required for telomerase action. In addition, normal telomere length can be restored by expressing a Cdc13-Est1p hybrid protein. These findings indicate that RPA activates telomerase by loading Est1p onto telomeres during S phase. We propose a model of in vivo telomerase action that involves synergistic action of RPA and Cdc13p at the G-rich 3' overhang of telomeric DNA.