The triplex formation between a 21nt oligodeoxyribonucleotide G₃TG₂TGT₂G₅TG₂TGT(CP1) and core promoter(Cp) fragment of hepatitis B virus(HBV) had a good specificity and stability, which was demonstrated by electrophoretic mobility shift analysis and DNaseⅠ footprinting experiment. Gel retardation assay showed CP1 could inhibit a specific cellular factor binding to Cp fragment in rat liver nuclear extracts. No inhibition of factor binding was observed by oligodeoxyribonucleotide CP3(TGTG₂TG₅T₂GTG₂TG₃), which could not form triplex with Cp fragment. These results indicate that specific repression of gene transcription of HBV DNA may be possible by triplex-formation.