For therapeutic or diagnostic use of monoclonal antibodies in clinical oncology, high-affinity IgG antibodies to tumor-associated antigens have to be generated. In order to find out by what immunization schedule the chance to generate such antibodies is increased, we evaluated three different immunization protocols with and without attempts to induce tolerance to common tissue antigens. Mice were immunized either (1) by repeated intraperitoneal injections, (2) by a single intrasplenic injection, or (3) by an intraperitoneal injection followed by an intrasplenic booster. Whereas a single intrasplenic immunization resulted in low-affinity antibodies to tumor-associated antigen, high-affinity antibodies were generated with the other two protocols, although at a lower frequency. No benefit was seen from tolerance induction. The intraperitoneal/intrasplenic protocol was found to be superior over the other protocols because of minimal antigen dose and immunization time, as well as a higher frequency of hybridoma formation.