Toll-like receptors (TLRs) detect the presence of microbial challenge and initiate innate immune defensive responses. In this work we have explored the mechanism and role of TLR dimerization in signal transduction using the newly developed technique of beta-lactamase protein fragment complementation, among others. We observed that TLR4 interactions with itself, with MyD88, or with TLR2 were accurately reported by the enzyme complementation technique. That technique, as well as co-immunoprecipitation, transfection-initiated cell activation, and site-directed mutagenesis all suggest an important role for TLR intracellular domains in receptor dimerization. These findings broaden our understanding of TLR self-interactions as well as heterodimer formation.