Two novel immunization methods (intrasplenic and intra-inguinal lymph node) have been developed for the production of polyclonal and monoclonal antibodies in mice. Freund's complete adjuvant and antigen were mixed in the ratio of 1:2 (v/v). Various concentrations of human serum albumin (HSA) were used as antigen. No primary immune response was induced with 0.1 microgram of HSA in either of the methods studied. Intrasplenic immunization resulted in the strongest primary immune responses using all other doses of HSA. The primary immune response induced by intrasplenic immunization with 0.5 microgram of HSA was higher than any response induced by subcutaneous immunization with various doses of HSA. Inguinal lymph node immunization was less effective than intrasplenic immunization but better than subcutaneous immunization with 1-50 micrograms of HSA. Comparisons were also made of the efficacy of different adjuvants when inducing primary immune responses with 1 microgram of HSA. Freund's complete adjuvant resulted in a much stronger response than Freund's incomplete adjuvant and alum. Both intrasplenic and inguinal lymph node immunization using 1-5 micrograms of HSA were able to induce strong primary immune responses. Secondary immunization with either method or intravenous injection 3 days before fusion resulted in a higher frequency of specific monoclonal antibodies.