We herein analyzed the regulation of phosphatidylinositol 3-kinase (PI 3-kinase) activity by CR2 activated on B lymphocyte cell surface. We demonstrated that CR2 activation triggered in vivo PI 3-kinase activity and interaction of PI 3-kinase p85 subunit with a tyrosine-phosphorylated p95 component. The specificity of PI 3-kinase activity was controlled using wortmannin and LY294002. CR2 activation did not trigger tyrosine phosphorylation of PI 3-kinase p85 subunit, but induced direct interaction of tyrosine phosphorylated p95 with the Src homology 2 domain of p85 subunit, as shown using glutathione-S-transferase fusion proteins. Despite identical molecular masses, immunoblotting analysis demonstrated that tyrosine-phosphorylated p95 that interacted in vivo and in vitro with p85 was neither CD19, the 95-kDa proto-oncogene vav, nor Gab1 (a 95-kDa adaptor molecule). Furthermore, p95 tyrosine phosphoprotein also expressed in K562A cells (CR2+ CD19- cells) interacted with Src homology 2 domain of PI 3-kinase p85 subunit after CR2 activation. Activated CR2 did not interact directly with p85 subunit or tyrosine-phosphorylated p95. This suggests the presence of an intermediate molecule between activated CR2 and tyrosine-phosphorylated p95, which may be 3BP2. In addition, in contrast to CD19 activation, CR2 activation did not trigger interaction of CD19 or Vav with PI 3-kinase p85 subunit or coprecipitation of PI 3-kinase activity with CD19. Together, these data clearly demonstrated that CR2 activation triggered in vivo PI 3-kinase activation through a pathway distinct from that triggered through CD19 activation.