Hepatitis delta antigens (HDAgs) are important for the replication and assembly of hepatitis delta virus (HDV). To understand the association between HDAgs and cellular proteins and the mechanism of viral multiplication, we have studied the interaction between HDAgs and nucleolin, a major nucleolar phosphoprotein. The interaction between HDAgs and nucleolin was first demonstrated by immunofluorescence staining studies. HDAgs and endogenous nucleolin were colocalized in the nucleoli of cultured cells transfected with plasmids encoding the small and large HDAg. Coimmunoprecipitation results indicated that the NH2-terminal domain of HDAg was essential for its binding to nucleolin. In vitro ligand binding assays revealed two nucleolin binding sites, NBS1 and NBS2. Each spanned amino acid residues 35-50 and 51-65, respectively, with a conserved core sequence K(K/R)XK. HDV replication was modulated by exogenous human nucleolin. In addition, a small HDAg mutant S-d65/75, which possesses both NBS1 and NBS2, was capable of transactivating HDV replication, whereas the small HDAg mutant S-d50/75, which retained NBS1 but not NBS2, was unable to support the replication of HDV. Thus, the nucleolin binding activity of HDAg is critical for its nucleolar targeting and is involved in the modulation of HDV replication.