Two immunization techniques that enable production of mouse monoclonal antibodies were evaluated in terms of small quantities of antigen. Various amounts of purified influenza A virus particles were applied either for in vitro sensitization in cultured splenocytes or for intrasplenic immunization, followed by hybridization of the immunized cells with mouse myeloma cells. Hybridomas producing specific antibodies for influenza viral proteins were detected by enzyme-linked immunosorbent assay when more than 50 micrograms of antigens was used for the in vitro immunization method, and at least 5 micrograms was necessary for a single intrasplenic immunization. On the other hand, as little as 60 ng of antigen administered in two intrasplenic injections was sufficient to produce specific hybridomas. Two out of six randomly selected monoclonal antibodies obtained using the repeated intrasplenic immunization method were IgG and the other four were IgM. Immunoprecipitation analyses revealed that the recognized antigens involved a viral inner protein (nucleocapsid protein), as well as an envelope glycoprotein (hemagglutinin). We conclude that immunization by two direct injections of antigen into the spleen is the most effective method for sensitization with nanogram quantities of insoluble antigen such as influenza viruses.