Expression of the c-myc protooncogene is estrogen regulated in estrogen receptor (ER) positive, hormone-dependent human breast cancer cells, but it is constitutively active in ER negative, hormone-independent breast cancer cells. To determine whether these differences are reflected in c-myc chromatin, DNase I hypersensitive sites (DHS) were mapped. Six DHS were detected in all cell lines studied, with DHS 3(2) being more prominent than DHS 3(1). The accessibility of DHS 2 was markedly greater in ER negative cells than in ER positive cells, and this relative accessibility remained unchanged when cells were grown in estrogen free medium. DHS 2, 3(1) and 3(2) map near the P0, P1 and P2 promoters, respectively. An analysis of promoter usage demonstrated that P2 was the preferred promoter. Thus, the differences in the accessibility of DHS 2 in c-myc chromatin of ER positive and negative cells likely reflects alterations in DNA-protein interactions in this region.