The aim of this study was to determine whether stimulation of toll-like receptor 2 (TLR2), TLR4, and TLR6 by their specific ligands induces the production of tumor necrosis factor-alpha (TNF-alpha) in fibroblast-like synoviocytes (FLS) from interleukin-1 receptor antagonist (IL-1Ra)-deficient mice. FLS were isolated from synovial tissues from IL-1Ra-deficient mice and stimulated with various ligands of TLRs. The concentrations of TNF-alpha, interleukin (IL)-1beta, and IL-10 in the culture supernatants of spleen cells were measured by ELISA, and mRNA levels were assessed by real-time PCR. The expression of TLR2, TLR4, TLR6, and TNF-alpha in the synovial tissue was quantified by immunohistochemistry. Cytokine production and TLR expression were measured in FLS stimulated in the presence of the TLR2 ligand PAM3, the TLR4 ligand lipopolysaccharide (LPS), and the TLR6 ligand zymosan, with and without blocking antibody to TNF-alpha and IL-1beta. Stimulation of TLR2, TLR4, and TLR6 by their specific ligands increased the production of TNF-alpha in FLS from IL-1Ra-deficient mice. The stimulatory effect of these TLR ligands showed a dose-dependent pattern. The combination of TLR2, TLR4, and TLR6 synergistically increased the production of TNF-alpha, IL-1beta, TLR2, TLR4, and TLR6. Addition of blocking antibodies to TNF-alpha and IL-1beta abrogated the stimulatory effect of the ligands of TLR2, TLR4, and TLR6 on the production of TNF-alpha, IL-1beta, TLR2, TLR4, and TLR6. These data show that TLR2, TLR4, and TLR6 ligation synergistically stimulates the production of TNF-alpha and IL-1beta in IL-1Ra-deficient mice and suggest that TLRs contribute to the perpetuation of spontaneous arthritis in this animal model.