Abstract B cells express the endosomal, DNA sensing, Toll-like receptor 9 (TLR9) that, in response to CpG oligonucleotides, signals through the adaptor protein MyD88 and activates the NF-kB pathway leading to the transcriptional activation of proinflammatory cytokines. In dendritic cells TLR9-MyD88 signaling is bifurcated into two distinct pathways, the NF-kB pathway and an IRF7-dependent pathway that results in the activation of type 1 interferon (IFN) genes. In these cells, monomeric CpG-B activates the NF-kB-dependent pathway and multimeric CpG-A or CpG-A coupled to the liposomal transfection reagent, Dotap (CpG-A-Dotap), activates the IRF-7-dependent pathway. Here we provide evidence that the B cell TLR9 signaling pathway is similarly bifurcated. Both CpG-B and CpG-B-Dotap potently activate the NF-kB-dependent pathway leading to IL-6 synthesis but neither trigger an IFN response. In contrast, CpG-A-Dotap but not CpG-A alone stimulates an IFN response as shown by increases in the levels of mRNA encoding IFN and in the amount of IFN protein produced and also by the activation of B cells from IFN-reporter mice both in vitro and in vivo. By confocal microscopy CpG-A and CpG-B appear to traffic to different intracellular vesicles. However, the cellular machinery that mediates the IL-6 and IFN pathways seems to be linked in that pretreatment of B cells with CpG-B blocks the subsequent activation of the IFN pathway by CpG-A-Dotap. It will be of interest to determine if natural DNA containing ligands might trigger the two pathways in B cells.