Toll-like receptors (TLRs), a family of transmembrane receptors, mediate pathogen recognition and activation of the human innate immune response. TLR signaling results in the production of proinflammatory products such as tumor necrosis factor α (TNFα). Recent findings have identified amyloid beta peptide (Aβ), the primary component of senile plaques in Alzheimer′s disease (AD), as a probable endogenous TLR ligand. Aggregated Aβ also has proinflammatory properties, thus our study investigated the role of TLRs in the Aβ-mediated inflammatory response using THP-1 human monocyte cells. THP-1 cells express most TLRs and secrete TNFα in response to both bacterial lipopolysaccharide (LPS) and Aβ. Our initial data confirmed a high sensitivity of these cells to TLR ligands LPS and Pam₃CSK₄, with EC₅₀ values of 117 ng/ml and 302 pg/ml, respectively. These cells were also sensitive to pre-assembled Aβ(1-42), the most pathogenic form of Aβ. To determine whether TLRs mediate cellular TNFα production by these molecules, we established a TLR antibody neutralization assay in which receptor blockade inhibits cell responsiveness to TLR ligands. The Pam₃CSK₄ and LPS responses were almost completely abolished by TLR2 and TLR4 antibodies, respectively. CD14, a ligand-binding TLR2/TLR4 accessory protein neutralized both ligand responses. We tested the antibody neutralization assay prior to Aβ treatment and found that TLR4, TLR2 and CD14 antibodies reduced the Aβ(1-42) response to 39%, 32%, and 56% of control, respectively. These results demonstrate that LPS (TLR4/CD14) and Pam₃CSK₄ (TLR2/CD14) stimulated TNFα production via TLRs and indicate that these receptors may also mediate an Aβ-induced innate immune response.