The small, 195-amino-acid form of the hepatitis delta virus (HDV) antigen (deltaAg-S) is essential for genome replication, i.e., for the transcription, processing, and accumulation of HDV RNAs. To better understand this requirement, we used purified recombinant deltaAg-S and HDV RNA synthesized in vitro to assemble high-molecular-weight ribonucleoprotein (RNP) structures. After transfection of these RNPs into human cells, we detected HDV genome replication, as assayed by Northern analysis or immunofluorescence microscopy. Our interpretation is that the input deltaAg-S is necessary for the RNA to undergo limited amounts of RNA-directed RNA synthesis, RNA processing, and mRNA formation, leading to de novo translation of deltaAg-S. It is this second source of deltaAg-S which then goes on to support genome replication. This assay made it possible to manipulate in vitro the composition of the RNP and then test in vivo the ability of the complex to initiate RNA-directed RNA synthesis and go on to achieve genome replication. For example, both genomic and antigenomic linear RNAs were acceptable. Substitution for deltaAg-S with truncated or modified forms of the deltaAg, and even with HIV nucleocapsid protein and polylysine, was unacceptable; the exception was a form of deltaAg-S with six histidines added at the C terminus. We expect that further in vitro modifications of these RNP complexes should help define the in vivo requirements for what we define as the initiation of HDV genome replication.