The repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genome integrity. The first step in DSB repair by homologous recombination is the processing of the ends by one of two resection pathways, executed by the Saccharomyces cerevisiae Exo1 and Sgs1–Dna2 machineries. Here we report in vitro and in vivo studies that characterize the impact of chromatin on each resection pathway. We find that efficient resection by the Sgs1–Dna2–dependent machinery requires a nucleosome-free gap adjacent to the DSB. Resection by Exo1 is blocked by nucleosomes, and processing activity can be partially restored by removal of the H2A–H2B dimers. Our study also supports a role for the dynamic incorporation of the H2A.Z histone variant in Exo1 processing, and it further suggests that the two resection pathways require distinct chromatin remodeling events to navigate chromatin structure.