Toll-like receptor (TLR) 4/MD-2 dimerization is thought to be required for the initiation of signaling during innate immune responses. In this study, we examined the molecular mechanisms underlying receptor dimerization in the context of accessory molecules, i.e. CD14 and lipopolysaccharide-binding protein (LBP), to determine whether dimerization is required for the initiation of signaling in response to LPS stimulation. We found that LPS-induced TLR4/MD-2 dimerization occurred only in membrane-associated CD14 (mCD14)-expressing cells. Furthermore, dimerization required LBP, but not soluble CD14 (sCD14), as an essential serum component. LPS-induced signaling as assessed by IkappaB-alpha degradation, however, occurred in mCD14-negative cells in the presence of serum and sCD14. Signaling also occurred in mCD14-positive cells in the absence of serum. Time course studies on mCD14-positive cells have demonstrated that LPS stimulation induces rapid activation of nuclear factor-kappaB and p38 in the presence of LBP (TLR4/MD-2 receptor dimerization) as compared with stimulation without LBP (receptor non-dimerization). This early activation was blocked by inhibitory anti-CD14 mAb. These studies suggest that LPS-induced TLR4/MD-2 receptor dimerization is not essential for signaling but prompts rapid signaling during innate immune responses.