A novel method of preparing hybridomas producing rat monoclonal antibodies was established. The enlarged medial iliac lymph nodes from rats injected via hind footpads with an emulsion of antigen and Freund's adjuvant were used for cell fusion. Ovalbumin was used as a representative antigen. The incidence of hybridomas producing antibody of interest with this method was about 10 times higher than that of hybridomas with the conventional method using mouse or rat spleen cells. The average percentages of hybridomas producing IgG1, IgG2a, IgG2b and IgG2c were 37.1%, 47.0%, 15.9% and 0.0%, respectively. A single injection with antigen was sufficient for immunization in this method.