To understand further the roles that negative regulatory signals may play in B cell immune responses, we compared three inhibitors of B cell proliferation: cross-linking CD19 with monoclonal antibody (mAb), signaling through Fc receptors by intact anti-mu mAb, and transforming growth factor-beta (TGF-beta). Each agent was tested for its ability to block proliferation and specific activation events induced in human tonsilar B cells activated by either cross-linking surface immunoglobulin, signaling through CD20, or direct activation of protein kinase C (PKC) by phorbol 12-myristate 13-acetate. We found that each inhibitor was functionally distinct. Both anti-CD19 mAb and anti-mu mAb inhibited anti-immunoglobulin activated cells and anti-CD20-activated cells, but neither inhibited cells activated by phorbol 12-myristate 13-acetate. TGF-beta, on the other hand, inhibited equally profoundly cells activated by each of the three regimens. These results suggest that TGF-beta blocks B cell activation at a step following the activation of PKC, whereas both signaling through CD19 and Fc receptor block early steps in the PKC activation pathway. Signaling through anti-CD19 mAb was unique in that proliferation of anti-immunoglobulin-activated cells was reduced on day 3 and then augmented subsequently. With all other inhibitory combinations the block was permanent. We conclude that each of these three inhibitors has unique important functions and therefore suggest that the effectiveness of negative signaling in B cell immune regulation will depend on the combinations of specific inhibitors modulating a specific activation program.