Bruton's tyrosine kinase (Btk) is known to be involved in a broad array of signal transduction pathways necessary for the proliferation, survival and development of B cells. Mutations in the Btk gene are causually linked to the development of X-linked agammaglobulinemia (XLA) in humans. We show here, that CD40 ligation leads to tyrosine phosphorylation of Btk in the human immature B cell line MHH-PREB-1. Even though the CD40-mediated tyrosine phosphorylation of Btk is less efficient than that mediated through the B cell receptor, CD40 stimulation results in a marked and continous activation of Btk in MHH-PREB-1 cells and, to a lesser extent, in a transient Btk activation in Daudi cells. Furthermore, in Daudi cells we observed that tyrosine phosphorylation and activation of Btk is correlated with its translocation from the cytosolic to the membraneous fraction. These data suggest that, in addition to the BCR, CD40 is a surface receptor through which the activity of Btk can be stimulated in human B cells.