In the early stage of infection, Sendai virus delivers its genome into the cytoplasm by fusing the viral envelope with the cell membrane. Although the adsorption of virus particles to cell surface receptors has been characterized in detail, the ensuing complex process that leads to the fusion between the lipid bilayers remains mostly obscure. In the present study, we identified and characterized cell lines with a defect in the Sendai virus-mediated membrane fusion, using fusion-mediated delivery of fragment A of diphtheria toxin as an index. These cells, persistently infected with the temperature-sensitive variant Sendai virus, had primary viral receptors indistinguishable in number and affinity from those of parental susceptible cells. However, they proved to be thoroughly defective in the Sendai virus-mediated membrane fusion. We also found that viral HN protein expressed in the defective cells was responsible for the interference with membrane fusion. These results suggested the presence of a previously uncharacterized, HN-dependent intermediate stage in the Sendai virusmediated membrane fusion. Sendai virus (SV)1 belongs to the Paramyxoviridae, carrying a nonsegmented, negative strand genomic RNA (15, 384 nucleotides). The viral envelope, made up of a lipid bilayer and two glycoproteins (F and HN), encapsulates the nucleocapsid, consisting of the genomic RNA and three nucleocapsid proteins (NP, P, and L) (1). In the early stage of infection, SV delivers the nucleocapsid into the cytoplasm by fusing its envelope with the cell membrane at neutral pH. SV also induces cell-to-cell fusion under appropriate conditions. These phenomena have been investigated intensively as a model of fusion between biological membranes (2).SV-mediated membrane fusion consists of two distinguishable stages: the binding of the viral envelope to the cell membrane, and the subsequent fusion between the lipid bilayers. The former stage, mediated by the interaction between viral HN protein and the cell surface receptors containing sialic acid (N-acetylneuraminic acid), is essential for subsequent membrane fusion (3, 4). A number of molecules have been assigned to SV receptors, including glycoproteins (5-9) and gangliosides (3, 10 -15). In cultured mammalian cells, gangliosides with terminal sialic acid residues, such as GD1a, GT1b, and GQ1b, have been reported to serve as functional receptors (10, 11).The second stage of fusion between the lipid bilayers is triggered by the viral F protein. This protein is initially synthesized as a precursor form (F 0 ), which is then processed by proteolytic cleavage to a mature form consisting of two polypeptides (F 1 and F 2 ) (16). The cleavage exposes a well conserved hydrophobic domain at the N terminus of the F 1 subunit (17), which binds cholesterol (18) and plays a principal role in the fusion between lipid bilayers. This simple two-step model has been thought to represent most types of membrane fusion induced by various envelope viruses (19). However, the recent finding that human immunodefici...