To investigate involvement of cellular glycosphingolipids in the propagation of influenza viruses in host cells, MDCK cells were treated with inhibitors for sphingolipid biosynthesis, fumonisin B1 and d,l-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol. Continuous treatment of the cells with either inhibitor during pre- and post viral inoculation, but not the pretreatment alone, significantly reduced viral infection, but not viral attachment to the cells. Immunocytochemical analysis demonstrated that cellular distribution of hemagglutinin, a viral glycoprotein, was drastically altered when the cells were continuously treated with the inhibitors during pre- and post viral inoculation, but not the pretreatment alone. Our findings strongly suggest that cellular sphingolipids play important roles in the events after viral adsorption to the host cells.