Activation of resting B cells requires an initial triggering of the B cell antigen receptor (BCR) and secondary stimuli through various cytokine receptors and B cell activation molecules including CD40. We found that activation of B cells through CD40 is selectively inhibited by an immunosuppressant drug, rapamycin. This effect of rapamycin on anti-CD40-mediated activation of B cells was observed using three different in vitro assays. Rapamycin suppressed the anti-CD40-induced proliferation of splenic B cells, suppressed differentiation to surface IgMhigh/IgDlow B cells, and inhibited an anti-CD40-mediated prevention of apoptosis induced by BCR cross-linkage of WEHI-231 cells. We next examined several known CD40 signal transduction pathways to identify the target of rapamycin in stimulated B cells. Rapamycin did not inhibit the activation of c-Jun N-terminal kinases (JNKs) induced by anti-CD40 stimulation nor the activation of immediate nuclear transcription factors of NF-kappaB. Therefore, rapamycin affects a novel element of the CD40 signal transduction pathway which influences the proliferation, differentiation, and prevention of apoptosis of B cells.