Nuclease S1-hypersensitive sites in a 40-kb region of the chicken genome including the domain of the alpha-globin genes were mapped. Brief treatment of isolated chicken erythroid cell nuclei with nuclease S1 allowed separation of an approximately 20-kb genomic DNA fragment containing the whole alpha-globin gene cluster. No S1-hypersensitive sites were observed in the internal part of the domain. The upstream S1 site was found in a DNA fragment of 1.7 kb where the origin of replication and several protein binding sites were identified previously. Precise mapping of the positions of S1 cleavage in this fragment and "in vivo" footprinting of DNA-protein interactions in isolated nuclei showed a correspondence with some of these protein binding sites. The possible significance of all these observations is discussed in connection with the replication origin and the nuclear matrix attachment regions in the framing structures.