Mitogenic reactivity, unit gravity sedimentation, surface membrane phenotype, and in vivo booster immunization have been used to delineate B cell subsets that secrete anti-tetanus toxoid antibody (Ig-Tet) in vitro. These subsets were characterized by their in vitro reactivity to pokeweed mitogen (PWM), bacterial lipopolysaccharide (LPS), tuberculin purified protein derivative (PPD), or on the basis of their spontaneous antibody secretion (lymphoblastoid cells). Five to 7 days after in vivo booster immunization with tetanus toxoid, lymphoblastoid cells, bearing the phenotype Ig-CR-FcγR-Ia+, were detected in the circulation and produced IgG-Tet without T cell or mitogen stimulation during a 3-day culture period. These cells were consistently the fastest sedimenting B cell subset observed, ranging from 6 to 12 mm/hr. These cells also showed considerable size heterogeneity and required DNA synthesis for maximum antibody production. IgM anti-tetanus toxoid antibody (IgM-Tet) synthesis was detected in B cells cultured with PWM-stimulated T cells and was not dependent on recent in vivo booster immunization. The majority of IgM-Tet synthesis was by cells bearing the phenotype μ+δ+FcγR ± Ia+. By 12 days after immunization, IgG-Tet synthesis was only detected in PWM- or PPD-stimulated B and T cell cultures. The majority of IgG-Tet produced by PWM-reactive B cells had the phenotype Ig+CR+FcγR-Ia+, yet they were heterogeneous in their expression of μ and δ. PWM-stimulated IgG-Tet production occurred over a narrow range of sedimentation velocities (4 to 6 mm/hr) that were slightly faster than the majority of the B cells (2 to 4 mm/hr). Total immunoglobulin stimulated by PWM gave a similar profile, but faster sedimenting cells (8 to 12 mm/hr) were found in some donors. LPS primarily stimulated Ig production in cells that sedimented slightly faster than the majority of B cells. The surface phenotype of LPS-reactive cells was similar to the PWM-reactive cells, i.e., Ig+Cr+FcγR-Ia+. In contrast to PWM-stimulated cells, however, little IgG-Tet was detected by LPS stimulation of lymphocyte cultures of 2-wk immunized donors, suggesting that differences between LPS- and PWM-reactive cells exist in vivo. PPD stimulated at least 2 B cell subsets. One subset sedimented between 8 and 12 mm/hr and secreted IgG-Tet, whereas a 2nd was found primarily in the 2 to 6 mm/hr velocities and synthesized total IgG. The phenotype of PPD-induced B cells that synthesized the majority of IgM and IgG and IgG-Tet was μ+δ-CR+FcγR-. The absence of surface membrane IgD thus distinguished PPD-reactive from PWM-reactive IgG-Tet-producing cells.