Bacteroides fragilis, which is part of the normal intestinal flora, is a frequent cause of serious disease, especially in diabetic and surgical patients. In these conditions, B. fragilis lipopolysaccharide (LPS) is likely to play a major pathophysiologic role. B. fragilis LPS is structurally different from classical enterobacterial LPS, whose biological activities are mediated by Toll-like receptor 4 (TLR4) activation. The ability of B. fragilis LPS to activate TLR4 and TLR2 was investigated here, since evidence on this issue is scarce and controversial. Each of four different protein-free B. fragilis LPS preparations could induce interleukin-8 responses in cells cotransfected with TLR4/CD14/MD2 but not TLR4/CD14 alone. Two of the preparations also induced cytokine production in cells cotransfected with TLR2/CD14 or in peritoneal macrophages from TLR4 mutant C3H/HeJ mice. Both of these activities, however, were lost after repurification with a modified phenol reextraction procedure. Importantly, all preparations could induce endotoxic shock in TLR2-deficient mice, but not in TLR4 mutant C3H/HeJ mice. Consistent with these findings, anti-TLR4 and anti-CD14, but not anti-TLR2, antibodies could inhibit B. fragilis LPS-induced cytokine production in human monocytes. Collectively, these results indicate that B. fragilis LPS signals via a TLR4/CD14/MD2-dependent pathway, and it is unable to activate TLR2. Moreover, our data document the occurrence of TLR2-activating contaminants even in highly purified B. fragilis LPS preparations. This may explain earlier contradictory findings on the ability of B. fragilis LPS to activate cells in the absence of functional TLR4. These data may be useful to devise strategies to prevent the pathophysiologic changes observed during B. fragilis sepsis and to better understand structure-activity relationships of LPS.