Hepatitis delta virus (HDV) contains a viroid-like circular RNA that replicates via a double rolling circle replication mechanism. It is generally assumed that HDV RNA is synthesized and remains exclusively in the nucleus until being exported to the cytoplasm for virion assembly. Using a [32P]orthophosphate metabolic labeling procedure to study HDV RNA replication (T. B. Macnaughton, S. T. Shi, L. E. Modahl, and M. M. C. Lai. J. Virol. 76:3920-3927, 2002), we unexpectedly found that a significant amount of newly synthesized HDV RNA was detected in the cytoplasm. Surprisingly, Northern blot analysis revealed that the genomic-sense HDV RNA is present almost equally in both the nucleus and cytoplasm, whereas antigenomic HDV RNA was mostly retained in the nucleus, suggesting the specific and highly selective export of genomic HDV RNA. Kinetic studies showed that genomic HDV RNA was exported soon after synthesis. However, only the monomer and, to a lesser extent, the dimer HDV RNAs were exported to the cytoplasm; very little higher-molecular-weight HDV RNA species were detected in the cytoplasm. These results suggest that the cleavage and processing of HDV RNA may facilitate RNA export. The export of genomic HDV RNA was resistant to leptomycin B, indicating that a cell region maintenance 1 (Crm1)-independent pathway was involved. The large form of hepatitis delta antigen (L-HDAg), which is responsible for virus packaging, was not required for RNA export, as a mutant HDV RNA genome unable to synthesize L-HDAg was still exported. The proportions of genomic HDV RNA in the nucleus and cytoplasm remained relatively constant throughout replication, indicating that export of genomic HDV RNA occurred continuously. In contrast, while antigenomic HDV RNA was predominantly in the nucleus, there was a proportionally large fraction of antigenomic HDV RNA in the cytoplasm at early time points of RNA replication. These findings uncover a previously unrecognized presence of HDV RNA in the cytoplasm, which may have implications for viral RNA synthesis and packaging.