To identify the regions in the chicken c-myc promoter that are necessary for the binding of a nuclear trans-acting factor CTCF--the potential oncogene activator--we used a synthetic analog of the natural binding site that contains three correctly spaced CCCTC-repeats that are known to be involved in CTCF-binding. Gel retardation experiments failed to detect any CTCF-binding activity with this synthetic site. We conclude that GC-transversions made in the regions presumed to be invalid, do in fact interfere with the protein binding. The secondary structure analysis with S1-nuclease shows the presence of an unusual DNA conformation of the CTCF-binding site in the supercoiled plasmids, that can not be detected with the artificial construction. The precise mapping of S1 nuclease cleavage reveals several hypersensitive sites in the CCCTC-zone. Thus, an altered secondary structure may be functionally important for the protein recognition in vivo.