SignificanceThe repair of double-strand DNA breaks by homologous recombination is initiated by the nucleolytic resection of the 5'-terminated strand at the DNA break. Genetic work in Saccharomyces cerevisiae identified three DNA end resection pathways: Mre11-Rad50-Xrs2, Dna2-Sgs1-Top3-Rmi1, and Exo1. Here we investigated the relationship between the three nucleolytic complexes in vitro. With a focus on Exo1, we show that it is stimulated by the single-strand DNA-binding protein, RPA, and also by the Mre11-Rad50-Xrs2 complex. Furthermore, our analysis provides biochemical evidence for the view that Exo1 and Dna2-Sgs1-Top3-Rmi1 function downstream of Mre11-Rad50-Xrs2 as independent and mutually exclusive DNA end-processing pathways.