Meiotic double-strand DNA breaks (DSBs), the lesions that initiate meiotic recombination at the HIS4 recombination hot spot, occur in a region upstream of the coding sequence associated with multiple DNase I-hypersensitive sites. Mutations in transcription factors that lead to loss of the DSBs result in the loss of some but not all DNase I-hypersensitive sites in the upstream region. A meiosis-specific change in chromatin structure is detected in strains with the wild-type hot spot but not in strains with alterations that elevate or reduce hot spot activity. The position and intensity of micrococcal nuclease-hypersensitive sites correlate poorly with the sites of DSB formation.