A trimer of hepatitis delta virus (HDV) cDNA in a retrovirus expression vector was transfected into subclone of the PLC/PRF/5 human hepatoma cell line, and a stable cell line (H1 delta 9) was clonally selected that supported the synthesis of both genomic and antigenomic sense HDV RNA. The H1 delta 9 cell line also expressed hepatitis delta antigen (HDAg) in cell nuclei in three distinct morphological patterns, including patterns typically seen in HDV-infected livers. HDAg expression was restricted to the smaller (p24) of the two HDAg-associated polypeptides in early passages of the H1 delta 9 cell line, but continuous passage of the cells resulted in increasing of expression of the larger (p27) HDAg-specific polypeptide. Passage of the H1 delta 9 cells also led to sustained expression of monomeric HDV RNA and a reduction in the levels of dimeric- and trimeric-HDV RNA. This was accompanied by an attenuation of virus-related cytotoxicity which was a feature of early cell passage numbers. HDV RNA replication in these cells was resistant to actinomycin D suggesting that replication was not dependent on continued expression from the transfected HDV cDNA and thus was likely to be self-sustaining.