Double-strand DNA breaks (DSBs) occur at recombination hotspots during Saccharomyces cerevisiae meiosis and are thought to initiate exchange at these loci. Analysis of DSB sites in three regions of the yeast genome indicated that breaks occur at or near many potential transcription promoters and that DSBs initiate most, if not all, meiotic recombination. DSB sites displayed deoxyribonuclease I hypersensitivity in chromatin from mitotic and meiotic cells, and changes in chromatin structure produced parallel changes in the occurrence of DSBs. Thus, features of chromatin structure that are established before meiosis play a role in determining where meiotic recombination events initiate.