The large delta antigen (LDAg) of hepatitis D virus (HDV), which is similar to the small delta antigen (SDAg), except it has 19 additional amino acids and an isoprenylation signal at the C-terminus, is crucial for interacting with hepatitis B surface antigen (HBsAg) to form a mature virion of HDV. Previous studies indicated that the LDAg alone, but not SDAg, can interact with HBsAg to form an empty particle. However, no evidence yet shows whether the intermolecular interaction of LDAg is necessary for forming an empty HDV particle. By cotransfection of plasmids encoding deletion or isoprenylation-negative mutants of LDAg with a plasmid encoding HBsAg into human hepatoma cells, we demonstrated that (i) the isoprenylation-negative LDAg cannot be secreted, (ii) the coiled-coil domain-deleted LDAg retains the secretion capability, (iii) the isoprenylation-negative LDAg can neither cosecrete with isoprenylation-positive LDAg nor suppress its secretion, and (iv) an intermolecular interaction between LDAgs is unlikely required for secretion. A hypothetical model of empty HDV particle containing HBsAg with isoprenylated LDAgs, which are probably present in a singular form, was then proposed.