Antigen-specific B cells from the peripheral blood of immunized donors were isolated following rosette formation with antigen-coated immunomagnetic beads and were phenotypically and functionally characterized. B cells separated with tetanus toxin (TT)- or keyhole limpet hemocyanin (KLH)-coated beads produced significant amounts of specific IgM and IgG antibodies when they were transformed with Epstein-Barr virus or cultured with autologous T cells in the presence of a specific antigen. Cells isolated from peripheral blood mononuclear cells (PBMC) with TT-coated beads consisted mostly of B cells (88.7% CD20+). B cells detected among the rosetting cells were predominantly sIgM+ with low percentages of the other isotype-expressing B cells. The mean percentage of TT-specific B cells among adult donors was 0.34% of PBMC, and increased to up to 2% following a booster immunization with antigen. A similar increase in the number of rosetting B cells was also observed following in vitro culture of PBMC with antigen. These findings demonstrate that immunomagnetic bead selection serves as a specific and reliable approach in identifying and isolating antigen-specific B cells from human PBMC and provides a valuable method for studying T-B interactions in antigen-specific immune responses.