Bruton's tyrosine kinase (Btk) plays a critical role in B cell Ag receptor (BCR) signaling, as indicated by the X-linked immunodeficiency and X-linked agammaglobulinemia phenotypes of mice and men that express mutant forms of the kinase. Although Btk activity can be regulated by Src-family and Syk tyrosine kinases, and perhaps by phosphatidylinositol 3,4,5-trisphosphate, BCR-coupled signaling pathways leading to Btk activation are poorly understood. In view of previous findings that CD19 is involved in BCR-mediated phosphatidylinositol 3-kinase (PI3-K) activation, we assessed its role in Btk activation. Using a CD19 reconstituted myeloma model and CD19 gene-ablated animals we found that BCR-mediated Btk activation and phosphorylation are dependent on the expression of CD19, while BCR-mediated activation of Lyn and Syk is not. Wortmannin preincubation inhibited the BCR-mediated activation and phosphorylation of Btk. Btk activation was not rescued in the myeloma by expression of a CD19 mutant in which tyrosine residues previously shown to mediate CD19 interaction with PI3-K, Y484 and Y515, were changed to phenylalanine. Taken together, the data presented indicate that BCR aggregation-driven CD19 phosphorylation functions to promote Btk activation via recruitment and activation of PI3-K. Resultant phosphatidylinositol 3,4,5-trisphosphate probably functions to localize Btk for subsequent phosphorylation and activation by Src and Syk family kinases.