Activation of human B cells in vitro either by cross-linking of surface immunoglobulins (sIg) or by triggering CD40 antigen, in the presence of interleukin-10 (IL-10) and interleukin-2 (IL-2), may result in high levels of immunoglobulin secretion in vitro. We studied the combined effects of ligation of the B-cell receptor (BCR) and CD40 [with anti-CD40 monoclonal antibody (mAb)] on B-cell proliferation and production of human immunoglobulin. For this purpose highly purified splenic B cells were cultured with various combinations of anti-CD40 and IL-10/IL-2 or IL-4 in the presence of CD32-transfected L cells. Simultaneous cross-linking of the BCR was achieved by mAb held on CD32-L cells or Staphylococcus aureus (SA). We found that dual BCR and CD40 ligation with IL-10/IL-2 leads to reduced immunoglobulin G (IgG) secretion compared with B cells stimulated with either anti-CD40 and IL-10/IL-2, or compared with B cells stimulated with SA or anti-BCR mAb and IL-10/IL-2. Dual BCR and CD40 ligation with anti-immunoglobulin mAb (anti-kappa + anti-lambda light chains) but not with SA induced a similar reduction in IgM production. The reduced immunoglobulin secretion found during dual ligation is accompanied by increased proliferation. This was independent of cytokine stimulation but SA/CD40-induced proliferation was increased in the presence of IL-10/IL-2, although not with IL-4. The combination anti-kappa and anti-lambda with anti-CD40 showed a long-term suppression of IgG and IgM production (at least 14 days), while anti-kappa or anti-lambda alone, or SA, allowed a moderate recovery of immunoglobulin production by day 14. These results suggest that simultaneous B-cell antigen receptor cross-linking and CD40 engagement via CD40L on T cells induces strong initial proliferation. This may be followed later by antibody production depending on the strength of the BCR signal and the presence of the appropriate cytokines.