Human B cells and plasmacytoid dendritic cells constitutively express Toll-like receptor (TLR)9 and respond to TLR9 ligands, as evidenced by nuclear factor kappa B translocation and cytokine secretion. However, TLR9 expression on B lymphocytes appears to be dependent upon both the state of activation and differentiation of the B cell population. In the current study, TLR9 mRNA expression was evaluated in transformed human B cell lines and correlated with their response to CpG. Among the B cell lines, the Burkitt lymphoma-derived Namalwa line had the highest level of TLR9 expression, 20-fold greater than spleen. Following incubation with CpG oligonucleotide, Namalwa cells secreted increased amounts of TNF, IL-6, and IL-10 and expressed the costimulator molecules CD40, CD80, and CD86. These functional responses to TLR9 activation occurred with similar EC(50) values in the 30- to 60-nM range. These results suggest that the Namalwa Burkitt lymphoma line may serve as a useful cell-based assay for the detection of novel TLR9 agonists as well as a model to further explore the regulation of TLR9 expression and signaling.