The chicken c-myc 5'-flanking sequence has previously been shown to bind multiple proteins present in undifferentiated and differentiated red blood cells. In this report the protein binding to one specific region within a hypersensitive site approximately 200 base pairs upstream of the start of transcription has been analysed in detail. Using a combination of a modified agarose gel retardation assay with 0-phenanthroline-copper footprinting in situ, missing contact point and methylation interference techniques, two proteins were found to bind to overlapping sequences; within 186-230 bp upstream of the start of transcription. One protein resembles the transcription factor Spl, the other is a protein which binds to three regularly spaced repeats of the core sequence CCCTC. This CCCTC-binding factor was termed CTCF. It requires additional sequences outside the three recognition motifs for tight binding. CTCF was purified to near homogeneity by sequence-specific DNA chromatography. The approximate molecular weight of the CTCF was estimated to be 130 000. Removal of HO bp sequence binding both CTCF and Spl-like proteins leads to a 4 to 8-fold ｩｾ] ｲ･. ｳ･@ ｾｾ LLZ ｾｴｩｯｾ@ of stably ｴｲ. ｮｾﾭ fected c-myc fus10n] ｯｮｳｴｲﾮｴｳ GGGG ｦｩｩｾ@] ｨｾ･ ｫ･ ｮ@ embryomc ｦｩｾｴｳ L@ suggesting that the CTCF is likely to be one of multiple nUclear factors involved in the transcriptional regulation of the chicken c-myc gene.