Transcription and replication of hepatitis delta virus (HDV) RNA is thought to be performed by host RNA polymerase II. The mechanism which enables polymerase II to use RNA as a template is unclear. However, since extensive intramolecular complementarity allows HDV RNA to form a rod-shaped structure, it is possible that the mostly double-stranded HDV RNA may resemble double-stranded DNA in structure, and can thus be used by RNA polymerase II as a template. To investigate this possibility, we examined whether the cDNA counterpart of HDV RNA contains a promoter and thus can drive the transcription and replication of HDV RNA. Circularized monomers of HDV cDNA, when transfected into various cell lines, were found to generate both monomeric and dimeric forms of HDV RNA and hepatitis delta antigen at levels comparable to those generated with HDV cDNA multimers under the control of a SV40 late promoter, suggesting that HDV cDNA contains endogenous promoters. Using chloramphenicol acetyltransferase and human growth hormone as reporter genes, the specific promoter activity for the synthesis of antigenomic HDV RNA was localized to a 29-nucleotide region (nucleotides 1650-1679), although an additional 224-nucleotide upstream region was also necessary for maximum activity. Similarly, promoter activity for the synthesis of genomic RNA was localized to a 160-nucleotide region around position 1679 that overlapped with the antigenomic promoter region. Since these regions are in a highly conserved double-stranded region of HDV RNA, they may represent RNA promoters recognized by RNA polymerase II. This result also suggests a convenient method, using circularized monomer HDV cDNA, to study HDV RNA replication.