We describe here for the first time successful application of the hydroxyl radical technique for genomic footprinting. In combination with two complementary techniques, DNase I footprinting and dimethyl sulfate methylation protection, we have obtained a high-resolution map of the promoter region of the yeast HSP82 heat shock gene, which resides within a constitutive nuclease hypersensitive site. We find that irrespective of transcriptional state, basal or induced, only one of three putative heat shock elements, HSE1, and the TATA box are tightly bound by proteins, presumably heat shock factor (HSF) and TFIID, respectively. Whereas the HSE1-associated factor binds tightly within the major groove of DNA, as discerned by protection of guanine residues from methylation by dimethyl sulfate in intact cells, the TATA factor appears to bind principally to the sugar-phosphate backbone, as revealed by strong protection from hydroxyl radical cleavage in whole-cell lysates. In addition, while HSE1 is strongly footprinted by DNase I in lysates, the TATA box is only weakly footprinted. Strikingly, both elements are associated with marked distortion of the DNA double helix in chromatin. Protein binding to HSE1 appears to cause a non-B-conformation, on the basis of a local 12 base-pair periodicity of hydroxyl radical protection and the presence of multiple DNase I hyperreactive cleavages flanking HSE1, whose pattern changes following heat shock. Similarly, helix distortion is evident in the vicinity of the TATA box, since hydroxyl radical detects a lower strand-specific hypersensitive site at the dyad center of an adjacent polypurine tract. Finally, the absence of discernable modulation in the DNase I cleavage pattern argues against the presence of a specifically positioned nucleosome within the IISP82 promoter region.