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This report describes the formation of human hybridomas after in vitro immunization of tonsillar lymphocytes and fusion of the stimulated lymphocytes with a human lymphoblastoid cell line, WI-L279HF2. Lymphocytes were stimulated in vitro with sheep erythrocytes (SRBC) and polyclonal activators (PCA). By varying the duration of in vitro immunization before fusion and varying the PCA concentrations, the optimal conditions for cell fusion, hybrid cell growth, nonspecific immunoglobulin (Ig) secretion, and secretion of antibody to SRBC were determined. Lymphocytes cultured allogeneically at 5 X 10(6) cells per well in 24-well plates with SRBC and PCA (PWM, 1/10,000 dilution or LPS, 5 micrograms/ml) fused with the greatest efficiency. PWM-stimulated cultures fused on day 6 of in vitro stimulation yielded the greatest number of wells containing Ig-secreting hybridomas, whereas LPS-stimulated cultures produced more wells with hybridomas secreting Ig when fused on day 7 of stimulation. In vitro stimulation conducted in tissue culture flasks produced fewer wells with hybrid cell growth and Ig secretion. A fusion ratio of 1:1 (lymphocyte:WI-L2729HF2) yielded the most hybrid formation. Ig levels in wells with hybrid cell growth varied greatly, from less than 10 ng/ml up to 30 micrograms/ml. Ig of IgG, IgA, and IgM isotypes were produced. The addition of lymphocyte conditioned medium to in vitro immunization cultures does not improve the yield of Ig-secreting hybridomas. Hybrid cells secreting antibody to SRBC were cloned and analyzed for HLA specificities and chromosome numbers. Additionally hybridoma cells were formed after in vitro stimulation of peripheral blood lymphocytes (PBL) of normal individuals by fusion of the stimulated lymphocytes with WI-L2-729HF2. Hybridoma formation with PBL was less successful, but hybridoma cells secreting antibody to SRBC after in vitro stimulation were isolated.