The study investigates the role of TRIF-related adaptor molecule (TRAM) in the signaling pathways activated by Toll-like receptors (TLRs) TLR3 and TLR4. TRAM, like TRIF, activates interferon regulatory factors (IRF)-3, IRF-7, and NF-κB. Unlike TRIF, which functions in both TLR3 and TLR4 signaling, TRAM is specifically involved in the TLR4 pathway. TRAM interacts with TRIF, MyD88 adaptor-like protein (Mal)/TIRAP, and TLR4 but not with TLR3. These findings suggest that TRAM and TRIF both play a role in LPS-TLR4 signaling, regulating the MyD88-independent pathway during the innate immune response to lipopolysaccharide (LPS). The study also examines the functional relationship between TRAM and TRIF, finding that they cooperate in the IRF-3 activation pathway. Additionally, TRAM is shown to be essential for LPS signaling, while TRIF is required for signaling by both TLR3 and TLR4. These results highlight the unique roles of TRAM and TRIF in the TLR4 signaling pathway and their potential importance in the specificity of the immune response to LPS.The study investigates the role of TRIF-related adaptor molecule (TRAM) in the signaling pathways activated by Toll-like receptors (TLRs) TLR3 and TLR4. TRAM, like TRIF, activates interferon regulatory factors (IRF)-3, IRF-7, and NF-κB. Unlike TRIF, which functions in both TLR3 and TLR4 signaling, TRAM is specifically involved in the TLR4 pathway. TRAM interacts with TRIF, MyD88 adaptor-like protein (Mal)/TIRAP, and TLR4 but not with TLR3. These findings suggest that TRAM and TRIF both play a role in LPS-TLR4 signaling, regulating the MyD88-independent pathway during the innate immune response to lipopolysaccharide (LPS). The study also examines the functional relationship between TRAM and TRIF, finding that they cooperate in the IRF-3 activation pathway. Additionally, TRAM is shown to be essential for LPS signaling, while TRIF is required for signaling by both TLR3 and TLR4. These results highlight the unique roles of TRAM and TRIF in the TLR4 signaling pathway and their potential importance in the specificity of the immune response to LPS.