A DNase I hypersensitive site was detected in chromatin formed over a human hsp70 gene segment after amplification in COS7 cells. Deletion mutant analysis was used to evaluate the sequence requirements for this chromatin structure. Determinants sufficient to form the hypersensitive site are contained in a 280 base-pair sequence corresponding approximately to the region that is hypersensitive. Deletion of sequences from either end of this region resulted in reduced hypersensitivity, suggesting that multiple genetic elements contribute to the formation of this chromatin structure. As has been reported for other heat shock genes, the hypersensitive chromatin structure is present prior to heat treatment and does not change in intensity or position after heat shock, in spite of the fact that hsp70 gene expression is completely dependent on heat induction. Sequence requirements for hypersensitivity were generally similar to those for heat-induced gene expression when mutant plasmids were tested at low copy number (e.g. in HeLa cells or in COS cells without amplification); however, deletion of sequences between -223 and -162 with respect to the start of transcription abolished the hypersensitive site but had no effect on gene expression. A barrier to exonuclease III digestion was detected within this region (near an imperfect inverted repeat sequence centered at position -202), suggesting that proteins are tightly bound to the DNA at this location. © 1988.