Btk deficient (BtkM) mouse B-lymphocytes do not proliferate when stimulated with anti-immunoglobulin (anti-Ig) antibodies. In order to characterize the molecular basis of this unresponsiveness we have compared early signal transduction pathways triggered by ligating the B cell antigen receptor (BCR) of purified resting B cells from normal C57BL/6 (wild-type) and BtkM mice on C57BL/6 background. BCR-induced signalling events that occur during the first few minutes of activation, such as bulk tyrosine phosphorylations, mitogen-activated protein kinase (MAPK) activation, PI3-kinase dependent PKB/Akt kinase phosphorylation/activation and PLCgamma2 tyrosine phosphorylation are comparable in wild type and BtkM B cells. However, the initial extracellular calcium influx is reduced and the BCR-induced accumulation of phosphatidic acid (PA) display a more transient profile in the BtkM cells. BCR ligation did not induce detectable phosphatidyl-choline PLD activity, suggesting that the reduced PA is owing to a reduction in the phospho-inositide hydrolysis. These findings further support the notion that the proliferative defect of Btk deficient mouse B cells in response to anti-immunoglobulin stimulation stems from a failure to sustain phospholipase-dependent signalling.