In this study, we have analyzed the effects of the immunosuppressive agent rapamycin on the activation of highly purified normal human B lymphocytes. When the polyclonal activators Staphylococcus aureus (SA) and soluble CD40 ligand (CD40L) were used to stimulate B cells, rapamycin inhibited both interleukin 2 (IL2)-dependent and -independent proliferation, as well as IL2-dependent differentiation into antibody-secreting cells. Cell cycle analysis indicated that rapamycin inhibited the progression of SA+IL2-stimulated B cells past the mid-G1 phase of the cell cycle. To begin to identify rapamycin-sensitive signaling events essential for B cell activation, we examined the effects of rapamycin on p34cdc2 and p33cdk2 kinase activities. SA+IL2 stimulation induced the activation of both cyclin-dependent kinases. Of interest, rapamycin abrogated the activation of both p34cdc2 and p33cdk2. Our results indicate therefore that rapamycin inhibits a number of SA- and CD40L-inducible events that may be necessary for both entry into S phase and for permitting subsequent B cell differentiation. These studies emphasize the utility of this drug as a tool to begin to dissect the activation pathways utilized by human B cells, as well as to provide implications for the therapeutic use of rapamycin in vivo.