Cross-linking of surface immunoglobulin (sIg) by antibodies against IgM, IgG and IgD activates B cells and in some circumstances can induce cell proliferation. We studied the potential link between anti-Ig-induced changes in the cytosolic free Ca2+ concentration ([Ca2+]i), inositol phosphate production and the ability to induce cell proliferation in the presence or absence of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA). Anti-IgM, but not anti-IgD or anti-IgG, induced cell proliferation in the presence but not the absence of TPA. Each of the antibodies induced a rapid increase in [Ca2+]i which appeared to be due to release of Ca2+ from internal stores. This was followed by a sustained increase in [Ca2+]i, apparently due to Ca2+ uptake from the extracellular medium. Anti-IgD induced the greatest increase in [Ca2+]i, anti-IgM induced intermediate changes and anti-IgG the lowest change. Since inositol 1,3,5-trisphosphate (IP3) can release Ca2+ from internal stores, we tested the ability of each anti-Ig isotype to increase concentrations of IP3. In contrast to the change in [Ca2+]i and proliferation, anti-IgG induced the most significant increase in IP3 concentrations. Taken together these data indicate that changes in [Ca2+]i, inositol phosphate production and anti-Ig-induced human B cell proliferation are not directly linked. They also demonstrate that changes in [Ca2+]i, inositol phosphate production and activation of protein kinase C are not sufficient to induce proliferation of human B cells. It appears that anti-IgM induces an additional Ca2+-independent, inositol phosphate-independent and protein kinase C-independent activation signal which can collaborate with TPA to induce B cell proliferation. The molecular events involved in this signal remain to be identified.