Immunostimulatory CpG oligodeoxynucleotide (CpG-ODN) sequences are known to directly activate B cells. We investigated the expression of the CpG receptor, Toll-like receptor 9 (TLR9), in human tonsil B cells, and determined functional responses following stimulation by a well-characterized stimulatory CpG-containing ODN sequence in the human immune system, ODN 2006. Tonsil B cells were found to express high amounts of TLR9 mRNA and protein, and exposure of B cells to CpG-ODN but not to an inactive control ODN induced a concentration- and time-dependent up-regulation of the activation markers CD23, CD25, CD40, CD54, CD80, CD86 and HLA-DR. However, significant induction of proliferation and the release of IL-6, IL-10, IgG and IgM were only noted when B cells were co-incubated with irradiated CD40L-expressing CHO cells. Endogenous IL-10 was identified as a critical mediator of Ig production, whereas all activating effects were independent of IL-6. Further, CpG-ODN counteracted IgE production induced by IL-4. Collectively, these findings suggest a synergistic role of the TLR9/CD40 system and a critical role for the immunomodulatory cytokine IL-10 in the orchestration of CpG-ODN-induced responses in B lymphocytes.