Cluster of differentiation ([CD]) 19 antigens are B-cell-specific molecules expressed on virtually all human cells of the B-lymphocyte lineage except plasma cells. We produced a new anti-CD19 monoclonal antibody (McAb), CLB-CD19, that was used to study the role of CD19 molecules in B-cell activation. Anti-CD19 McAb induced mobilization of free intracellular calcium ([Ca2+]i) in Daudi cells, but not in normal spleen or tonsillar B cells, for which crosslinking with a second anti-mouse Ig antibody was not required. Anti-CD19 McAb inhibited B-cell proliferation induced by anti-IgM coupled to Sepharose beads. This inhibitory effect was overcome by the addition of nonmitogenic concentrations of phorbol myristate acetate. Anti-CD19 McAb did not interfere with Staphylococcus aureus- or B-cell growth factor-induced B-cell proliferation. Anti-CD19 McAb inhibited T-cell-dependent polyclonal B-cell differentiation in pokeweed mitogen-, interleukin 2-, or anti-CD3-driven culture systems. Delayed addition studies showed that once differentiation of B cells was induced, CD19 molecules lost their regulating function. Taken together, our results indicate that CD19 molecules play a regulatory role in B-cell proliferation and differentiation.