Bruton's tyrosine kinase (BTK) was previously demonstrated to be a mediator of oxidative stress-induced apoptosis in irradiated neoplastic B-cells and B-cell precursors. Defective BTK expression in leukaemic B-cell precursors from infants with t(4;11) acute lymphoblastic leukaemia has been associated with radiation resistance. The present study examined whether BTK mediates apoptosis during oxidative stress by interfering with the anti-apoptotic function of signal transducer and activator of transcription 3 (STAT3). BTK physically associated with and tyrosine phosphorylated STAT3; this association was promoted by pervanadate (PV)-induced oxidative stress. The BTK/STAT3 interaction appeared to prevent STAT3 response to oxidative stress, because PV-induced STAT3 activation was markedly enhanced in DT40 chicken lymphoma B-cells that were rendered BTK-deficient by targeted disruption of the btk gene as well as in BTK-deficient RAMOS-1 human lymphoma B-cells. These BTK-deficient cells were highly resistant to oxidative stress-induced apoptosis triggered by PV treatment. Reconstitution of BTK-deficient DT40 cells with wild-type human BTK gene eliminated the amplification of the STAT3 response and restored the PV-induced apoptotic signal. Similarly, while the BTK-positive NALM-6 human leukaemic B-cell precursor cell line showed no STAT3 activation after PV treatment and was exquisitely sensitive to PV-induced apoptosis, PV failed to induce apoptosis in BTK-deficient RAMOS-1 human lymphoma B-cells that showed a robust STAT3 response. These results provide unprecedented biochemical and genetic evidence for a unique mode of cross-talk that occurs between BTK and STAT3 pathways during oxidative stress, whereby BTK may trigger apoptosis via negative regulation of the anti-apoptotic STAT3 activity.