An influenza virosome vaccine, consisting of influenza hemagglutinin-neuraminidase antigens (HANA), the muramyldipeptide derivative 6-O-(2-tetradecylhexadecanoyl)-N-acetyl-muramyl-L-alanyl-D-isoglutamine (B30-MDP) and cholesterol, was prepared by the detergent removal method using a flow-through dialyzer, Liposomat. The shape of the influenza virosome vaccine mimicked that of the influenza virus, namely, B30-MDP/cholesterol vesicle covered with HANA spikes. The particle size and size distribution of prepared virosomes were evaluated by quasi-elastic laser light scattering and gel permeation chromatography. The state of incorporation of HANA. spikes at the membrane surface of virosome was confirmed by electron microscopy. The results indicated that virosome formation was influenced by initial BSO-MDP concentration in the mixed micellar solution prior to dialysis. It is apparent that the initial concentration of P30-MDP, relative to the mixed-micelle-to-virosome transition, is an important factor for virosome preparation. Accordingly, the transition was evaluated and clarified by the changes in membrane fluidity and particle size during the dialysis process. The results indicated that it is important to adjust the initial B30-MDP concentration in the mixed micellar solution before dialysis so that the concentration for the mixed-micelle-to-vesicle transition coincides with that for mixed-micelle-to-rosette transition appropriate for virosome formation.