The complementary DNA (cDNA) of hepatitis delta virus (HDV), constructed as tandem repeats under appropriate exogenous promoters, has been used to initiate viral replication in transfection experiments, Whether the structure of tandem repeats is essential has not yet been systematically examined. In this study, expression vectors containing only an HDV cDNA monomer, permutated at unique position of the genome, were shown still able to initiate viral replication. Furthermore, HDV cDNA monomer, separated from plasmid sequences by restriction enzyme digestion, also could be used to initiate viral RNA replication. The competence of HDV cDNA alone to direct viral RNA production suggested the presence of cryptic internal promoter-like elements. Such elements were actually demonstrated by the chloramphenicol acetyltransferase assay. Therefore, HDV cDNA, in contrast to that of viroids, could be used to initiate viral RNA replication in monomeric form. This observation simplified the use of HDV cDNA for studying viral biology in transfection systems.