Partially purified estrogen receptor prepared from heifer uterine cytosol, and labeled in vitro with tritiated estradiol, was used to locate receptor binding sites in target and non-target nuclei from various bovine tissues. Nuclei were digested to various extents with bovine pancreatic deoxyribonuclease I, micrococcal nuclease or endogenous nuclease and then assessed for their ability to bind charged estrogen receptor. After very brief digestion with DNAase I, such that only hypersensitive sites were cleaved, calf uterus nuclei were no longer able to bind estrogen receptor. Brief digests with micrococcal nuclease or endogenous nuclease, such that most DNA was still of polynucleosomal length, eliminated the binding ability of both calf and heifer uterus nuclei. These results suggest that estrogen receptor binds to pre-existing nuclease hypersensitive sites. Interestingly, nuclei digested by HaeIII restriction endonuclease, which cleaves at specific sequences, demonstrated no loss of labeled estrogen receptor binding, even though digestion products were of similar size to those obtained from nuclei after treatment with the other nucleases. Since nuclease hypersensitive sites occur in regulatory regions of actively transcribed genes, including estrogen-inducible genes, binding of estrogen receptor at these sites, in vivo, may be part of the mechanism by which transcription is induced.