Scavenger receptor associated with endothelial cells I (SREC-I) was shown to be expressed in immune cells and to play a role in the endocytosis of peptides and antigen presentation. As our previous studies indicated that SREC-I required intact Toll-like receptor 4 (TLR4) expression for its functions in tumor immunity, we examined potential interactions between these two receptors. We have shown here that SREC-I became associated with TLR4 on binding bacterial lipopolysaccharides (LPS) in RAW 264.7 and HEK 293 cells overexpressing these two receptors. The receptors then became internalized together in intracellular endosomes. SREC-I promoted TLR4-induced signal transduction through the NF-kB and MAP kinase pathways, leading to enhanced inflammatory cytokine release. Activation of inflammatory signaling through SREC-I/TLR4 complexes appeared to involve recruitment of the receptors into detergent-insoluble, cholesterol-rich lipid microdomains that contained the small GTPase Cdc42 and the non-receptor tyrosine kinase c-src. Under conditions of SREC-I activation by LPS, TLR4 activity required Cdc42 as well as cholesterol and actin polymerization for signaling through NF-kB and MAP kinase pathways in RAW 264.7 cells. SREC-I appeared to respond differently to another ligand, the molecular chaperone Hsp90 that, while triggering SREC-I-TLR4 binding caused only faint activation of the NF-kB pathway. Our experiments therefore indicated that SREC-I could bind LPS and might be involved in innate inflammatory immune responses to extracellular danger signals in RAW 264.7 cells or bone marrow-derived macrophages.