A variety of signals, mediated via either the B cell Ag receptor (BCR), or non-BCR molecules such as CD40 or cytokine receptors, have been shown to be crucial for the regulation of B cell survival, growth, and differentiation. Although it is clear that a variety of signaling pathways can be activated in B cells in a stimulus-dependent manner, it remains unknown whether differential activation of these signaling pathways is the underlying mechanism controlling B cell fate, i.e., growth vs differentiation. Initial studies reported here indicated that stimulation of highly purified peripheral blood B cells with the polyclonal B cell activators Staphylococcus aureus and CD40 ligand (CD40L) resulted in the rapid induction of phosphatidylinositol 3-kinase (PI 3-kinase) activity. Moreover, pretreatment of B cells with wortmannin, a specific inhibitor of PI 3-kinase, resulted in a complete block in induction of PI 3-kinase activity. The effects of wortmannin, as well as a second PI 3-kinase inhibitor, LY294002, on the induction of both B cell growth and differentiation were therefore investigated. Although these PI 3-kinase inhibitors variably inhibited B cell DNA synthesis in a stimulus-dependent manner, both drugs effected a near-complete block of the ability of each of these stimuli to induce Ig production. Furthermore, separation of B cells into naive IgD+ and postswitch IgD- B cells failed to reveal differential sensitivity of these populations to wortmannin. These results suggest that activation of PI 3-kinase, or other wortmannin- and LY294002-sensitive targets, is a crucial event that occurs during the differentiation of normal human B lymphocytes. The differential sensitivity of B cell responses to inhibitors of PI 3-kinase supports the notion that distinct signal transduction pathways are involved in differentiation vs proliferation of normal human B lymphocytes.