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 virus-mediated membrane fusion.