Toll-like receptor 4 (TLR4) is a signaling receptor for liwpopolysaccharide (LPS) but requires MD-2, a molecule associated with the extracellular TLR4 domain, to respond efficiently to LPS. The purpose of this study was to determine the critical stretch of primary sequence in the TLR4 region involved in MD-2 recognition. TLR4 and TLR4/2a chimera consisting of the TLR4 region Met(1)-Phe(54) and the TLR2 region Ala(53)-Ser(784) were coprecipitated with MD-2, but the deletion mutant TLR4(DeltaE24-P34) in which the TLR4 region Glu(24)-Pro(34) was deleted failed to coprecipitate. In agreement with the MD-2 binding, LPS-conjugated beads sedimented TLR4 and TLR4/2a chimera but not TLR2 with MD-2. TLR4(DeltaE24-P34) barely coprecipitated. with LPS-beads. The cells that had been cotransfected with TLR4(DeltaE24-P34) and MD-2 did not induce NF-kappaB activation in response to LPS. These results clearly demonstrate that the amino-terminal TLR4 region of Glu(24)-Pro(34) is critical for MD-2 binding and LPS signaling. (C) 2005 Elsevier Inc. All rights reserved.