TLRs sense pathogens and transmit intracellular signals via the use of specific adapter proteins. We designed a set of “blocking peptides”(BPs) comprised of the 14 aa that correspond to the sequences of the BB loops of the four known Toll-IL-1 resistance (TIR) domain-containing adapter proteins (ie, MyD88, TIR domain-containing adapter inducing IFN-β (TRIF), TRIF-related adapter molecule (TRAM), and TIR-domain containing adapter protein (TIRAP)) linked to the cell-penetrating segment of the antennapedia homeodomain. LPS (TLR4)-mediated gene expression, as well as MAPK and transcription factor activation associated with both MyD88-dependent and-independent signaling pathways, were disrupted by all four BPs (TRAM≈ MyD88> TRIF> TIRAP), but not by a control peptide. In contrast, none of the BPs inhibited TLR2-mediated activation of MAPKs. Only the MyD88 BP significantly blocked Pam3Cys-induced IL-1β mRNA; however, the inhibitory effect was much less than observed for LPS. Our data suggest that the interactions required for a fully functional TLR4 signaling “platform” are disrupted by these BPs, and that the adapter BB loops may serve distinct roles in TLR4 and TLR2 signalosome assembly.