We have recently identified a novel human B cell differentiation factor, 446-BCDF, derived from anti-CD3-stimulated peripheral blood (PB) T cells. This novel cytokine, which may act through a pertussis toxin-sensitive G(i)-linked receptor, induces a 5- to 100-fold increase in immunoglobulin (Ig) secretion by SAC (0.001%, v/v)-activated PB B cells. Coculture of B cells with 446 BCDF induces a decrease in intracellular cAMP which is necessary but not sufficient to drive terminal B cell differentiation. A second signal appears to be required. We therefore measured Ca2+ flux in indo-1 AM-loaded PB B cells. Stimulation with 446-BCDF resulted in an immediate rise in intracellular Ca2+ comparable to that seen with the anti-IgM mAb HB57, Ca2+ appeared to be mobilized from internal stores as pretreatment with BAPTA but not EGTA inhibited the response. Ca2+ mobilization was critical for the induction of differentiation as BAPTA pretreatment of PB B cells completely inhibited Ig secretion without affecting cell viability. In contrast, neither SAG, rIL6, IL2, IFN-γ, nor IL4 could mobilize Ca2+. Pertussis toxin, a G(i) and Go protein inhibitor, was able to inhibit 446-BCDF-induced Ca2+ flux as well as Ig secretion, To determine whether the Ca2+ flux was generated in the course of inositol phosphate turnover, we measured IP3 turnover and the translocation of PHC from cytosol to membrane, An increase in IP3 comparable to that seen with a monoclonal anti-human IgM antibody was noted and was specifically inhibited by the 446-BCDF-specific mAb 929. Interestingly, no membrane PKC was demonstrable in either SAC- or BCDF stimulated B cells, although PMA (50 ng/ml) could directly activate PKC. To confirm these findings functionally, B cells were stimulated with SAC and 446-BCDF in the presence of two known PKC inhibitors, staurosporin and calphostin. No inhibition of Ig secretion was detected at any concentration tested (0.39-100 nM staurosporin and 0.0625-1 μM calphostin C). These data suggest that induction of B cell differentiation is a Ca2-dependent and PTX-sensitive event. © 1995 Academic Press. All rights reserved.