To examine the role of hepatitis delta virus antigen in the replication of hepatitis delta virus RNA, we have transfected a stable HDAg-positive cell line (A3) and the parental HDAg-negative line (HepG2) with HDV RNA produced in vitro; synthesis of complementary HDV RNA was only detected in HDAg-positive cultures. In contrast, nuclear homogenates from both HDAg-positive and -negative cells synthesized comparable levels of complementary RNA from exogenous HDV RNA. These findings indicate that HDAg is not a necessary component of the transcriptional complex and suggest with other evidence, that a major role for HDAg is likely to be transport of HDV RNA from cytoplasm to nucleus. Transcription of HDV RNA by intact nuclei was sensitive to 1 microgram/ml alpha-amanatin providing firm evidence that, like viroids, this function is performed by host RNA polymerase II.