Myeloid differentiation factor MyD88 is the essential adaptor protein that integrates and transduces intracellular signals generated by multiple Toll-like receptors including receptor complex for interleukin (IL) 1beta, a key inflammatory cytokine. IL1beta receptor complex interacts with MyD88 via the Toll/IL1 receptor (TIR) domain. Here we report structure-function studies that help define the MyD88 TIR domain binding sites involved in IL1beta-induced protein-protein interactions. The MyD88 TIR domain, employed as a dominant negative inhibitor of IL1beta signaling to screen MyD88 TIR mutants, lost its suppressing activity upon truncation of its Box 3. Accordingly, mutations of Box 3 residues 285-286 reversed the dominant negative effect of the MyD88 TIR domain on IL1beta-induced and NFkappaB-dependent reporter gene activity and IL6 production. Moreover, mutations of residues 171 in helix alphaA, 195-197 in Box 2, and 275 in betaE-strand had similar functional effects. Strikingly, only mutations of residues 195-197 eliminated the TIR-TIR interaction of MyD88 and IL1 receptor accessory protein (IL1RAcP), whereas substitution of neighboring canonical Pro200 by His was without effect. Mutations in Box 2 and 3 prevented homotypic MyD88 oligomerization via TIR domain. Based on this structure-function analysis, a three-dimensional docking model of TIR-TIR interaction between MyD88 and IL1RAcP was developed.