Mycobacteria and their cell wall component lipoarabinomannan (LAM) have recently been established as agonists for TLR2. Our transfection studies with single and pairwise combinations of TLRs 1, 2, 6 and 10 reveal that only TLR1 and TLR2 together mediate strong activation of NF-kappaB-driven luciferase activity in response to LAM. Co-operative signaling by TLR1 and TLR2 is observed using either non-capped or mannose-capped LAM as a stimulus. Moreover, we have found that phosphatidylinositol mannosides, simple biosynthetic precursors of LAM, also activate cells through the combined actions of TLR1 and TLR2. Co-immunoprecipitation studies show that TLR1 and TLR2 are physically associated, independently of the presence of LAM. To address the mechanism of LAM-induced TLR activation we have used TLR fusion proteins in a protein fragment complementation assay. The results of this assay suggest that LAM alters the physical interaction between the intracellular signaling domains of TLR1 and TLR2. Together, these results identify LAM as an agonist for TLR1 and TLR2 and support the idea that LAM initiates transmembrane signaling by altering the physical association between TLR1 and TLR2.