The antigen-specific activation of murine nonimmunized B lymphocytes subsequently used in hybridization experiments has been investigated by using phylogenetically conserved antigens or autologous immunogens. This in vitro immunization was supported by B cell growth and differentiation factors derived from phorbol myristate acetate-stimulated EL-4 thymoma cells and mixed lymphocyte cultures (MLC). A filter immuno-plaque assay was used to evaluate the effect of different activation procedures on the number of antigen-specific plaque-forming cells (PFC). We first determined the requirement for MLC-derived lymphokines in the in vitro immunization. An optimal number of antigen-specific PFC was obtained when using 33 to 50% of the supernatant from a 48-hr MLC to support the activation. B cell growth and differentiation factors derived from EL-4 cultures were then tested for their abilities to potentiate the number of PFC by using both unseparated spleen cells and highly purified Ig-positive B cells as target cells. The combination of lymphokines found in supernatants from 25% EL-4 thymoma culture and 33% MLC yielded the highest number of PFC when used to support an in vitro immunization. This optimal factor preparation was used to determine the kinetics (4 to 7 days) and the dose response (0.01 to 10 micrograms antigen/ml) of antigen-specific B cell activation before using the immunized splenocytes as parental cells in cell fusion experiments. Mouse albumin and hemoglobin, actin (25 micrograms/ml), RNA polymerase II (5 micrograms/ml), as well as syngeneic mouse serum were used to immunize BALB/c spleen cells in vitro. We obtained antigen-specific PFC by using all of the different immunogens, including syngeneic mouse serum, and the in vitro immunized cells were then used in hybridization experiments. The specific efficiencies of each fusion that made use of cells immunized with mouse albumin, hemoglobin, syngeneic mouse serum, actin, or RNA polymerase II were 12, 31, 33, 52, and 22%, respectively, which illustrated the apparent lack of immune tolerance found when the immunization was performed in culture.