The 5' flanking region of the α-subunit gene of human chorionic gonadotropin was tested for the binding with the nuclear extracts of the JAR choriocarcinoma cells. Gel mobility shift assay has demonstrated that two BstNI restriction fragments, 150 bp and 340 bp respectively, have binding activities with the nuclear extracts. The 150 bp fragment was chosen to examine in detail, since several transcription factor binding sites have already been reported within the 340 bp fragment. Band competition assay showed that the binding of 150 bp fragment is highly sequence-specific. The binding activity was further narrowed down to a 55 bp fragment, which was generated by NsiI digestion of the 150 bp fragment. Dnasel footprinting assay revealed the binding sites on the nucleotide levels from -511 to -499. The binding sites were again confirmed by a oligonucleotide-directed mutagenesis of the 150 bp fragment followed by a gel mobility shift assay. Functional aspect of this binding was evaluated with the DNA-mediated gene transfer techniques. However, in several appoaches tried so far, no effect on the transcription efficiency was observed, implicating a structural role of the binding. In this line, it is worth to note that the binding activity of the 150 bp fragment was observed in every cell or tissue nuclear extracts tested so far