Monospecific, affinity-purified polyclonal antibodies reacting with the amino-terminal half of the mouse Toll-like receptor 4 (Tlr4) ectodomain failed to block LPS effects and, to the contrary, were capable of inducing TNF synthesis when applied to mouse macrophages and cross-linked with a secondary antibody. This effect was observed with macrophages derived from C3H/HeN and C57BL/10ScSn mice, but not with macrophages derived from C3H/HeJ or C57BL/10ScCr mice, indicating a specific, Tlr4-dependent effect. Neither primary nor secondary antibody caused any response if administered in the absence of the other reagent, nor was any response observed in cells from mice lacking Tlr4, or bearing the Lps(d) mutation of Tlr4. These findings support several conclusions. Tlr4, the essential transducer of LPS responses, may act independently of LPS itself. LPS needs not be internalized, nor must it bind to a secondary target within the cell in order to exert its effect; rather, the receptor alone is required for initiation of a signal. The data are consistent with the hypothesis that a conformational change in Tlr4 is required for activation via this receptor, and reveal that the amino-terminal half of the Tlr4 ectodomain is a target sufficient for antibody-mediated activation.