CD40 is a 50-kD glycoprotein that plays an important role in B cell survival, memory, and immunoglobulin isotype switch. Engagement of the CD40 antigen by monoclonal antibodies (mAbs) results in increased protein tyrosine kinase (PTK) activity, which plays an important role in mediating the biologic effects of CD40. We demonstrate, using an in situ phosphorylation technique, that CD40 cross-linking by the anti-CD40 mAb 626.1 resulted within 1 min in increased phosphorylation of the src type kinase, lyn, in Daudi B cell lines and remained sustained for up to 20 min. The activity of lyn kinase, as measured by immune complex kinase assay, was also increased after CD40 engagement, with similar kinetics. In contrast, the phosphorylation and activity of fyn, fgr, and lck kinases demonstrated minimal changes following stimulation of Daudi cells with mAb 626.1 over this same time period. CD40 engagement also resulted in phosphorylation of phospholipase C gamma 2 of phosphatidylinositol (PLC gamma 2) and phosphatidylinositol (PI)-3-kinase. Phosphorylation of PI-3-kinase was shown to be associated with an increase in its enzymatic activity. These results suggest that lyn plays an important role in CD40-mediated PTK activation and identify PLC gamma 2 and PI-3-kinase targets for CD40-mediated phosphorylation, suggesting a role for these two enzymes in CD40 signal transduction.