Synthetic oligonucleotides containing CpG motifs have been shown to induce proliferation, differentiation, and cytokine production in B cells, macrophages, and dendritic cells through a TLR9-dependent mechanism. A class (CpG-A) and B class (CpG-B) oligonucleotides display distinct physical properties. CpG-A, but not CpG-B, can multimerize to form exceedingly large lattices. CpG-A cannot effectively activate B cells but does induce plasmacytoid dendritic cells to produce high levels of IFNα, while CpG-B is a potent B cell mitogen. In this study, we report that CpG-A is internalized by B cells, and CpG-A and CpG-B accumulate in distinct intracellular compartments. When present in the form of an immune complex (CpG-A IC), CpG-A is taken up more efficiently by AM14 IgG2a-specific B cells, and elicits a robust TLR9-dependent B cell proliferative response. B cells proliferating comparably and in a TLR9-dependent fashion in response to CpG-A IC and CpG-B exhibited distinct cytokine profiles. CpG-A IC induced enhanced production of RANTES and markedly reduced levels of IL-6 when compared with CpG-B. We also found that engagement of the AM14 BCR by a protein IC, which cannot by itself induce proliferation, promoted TLR9-dependent but BCR-independent proliferation by bystander CpG-A or fragments of mammalian dsDNA. These data identify direct and indirect mechanisms by which BCR engagement facilitates access of exogenous ligands to TLR9-associated compartments and subsequent B cell activation. Copyright © 2009 by The American Association of Immunologists, Inc.