Two different immunization protocols using haptens 1 and 2 have been employed for the generation of catalytic antibodies capable of hydrolyzing eater 3. Three successive injections with one hapten 1 or 2, a protocol referred to as homologous immunization, provided hydrolytic antibodies with a rate acceleration in the range of 10(3)-10(4). These antibodies exhibited inhibitory activity only by the hapten used for the immunization. On the other hand, two successive injections with hapten 1 followed by a boost with hapten 2, a protocol referred to as heterologous immunization, induced catalytic antibodies with a rate acceleration up to 1.5 x 10(5). The majority of these catalytic antibodies possessed cross-reactivities to haptens 1 and 2, and the catalytic activities were effectively inhibited by both haptens. Control experiments have suggested that catalytic antibodies via heterologous immunization are derived through the unique stimulation of 1-primed memory B-cells by the secondary hapten 2, but not through the primary response of virgin B-cells by 2. Two catalytic antibodies H2-23 and H5H2-42, generated via homologous immunization with 2 and heterologous immunization with haptens 1 and 2, respectively, were selected for the detailed kinetic studies. Antibody H2-23 showed burst kinetic behavior and the burst phase was eliminated by the addition of p-nitrophenolate 5. Antibody H5H2-42 has no burst phase and exhibited high multiple turnover activity. The pH-dependent kinetic characterization of H5H2-42 suggested that bifunctional catalytic residues in the antibody combining site likely exist in the active site. These results imply that the heterologous immunization strategy offers a potential means of introducing multiple catalytic residues into antibody combining sites without recourse to complicated synthesis of multifunctional haptens.