Members of the Toll-like receptor (TLR) family are components of the mammalian anti-microbial response, signaling with a domain closely related to that of IL-1 receptors. In this report the expression and function of TLR1, a TLR of unknown function, are examined. TLR1 is expressed by monocytes, as demonstrated using a novel mAb. Monocytes also express TLR2. TLR1 transfection of HeLa cells, which express neither TLR1 nor TLR2, was not sufficient to confer responsiveness to several microbial extracts. However, cotransfection of TLR1 and TLR2 resulted in enhanced signaling by HeLa cells to soluble factors released from Neisseria meningitidis relative to the response with either TLR alone. This phenomenon was also seen with high concentrations of some preparations of LPS. The N. meningitidis factors recognized by TLR1/TLR2 were not released by N. meningitidis mutant in the LpxA gene. Although LpxA is required for LPS biosynthesis, because cooperation between TLR1 and TLR2 was not seen with all LPS preparations, the microbial component(s) TLR1/2 recognizes is likely to be a complex of LPS and other molecules or a compound metabolically and chemically related to LPS. The functional IL-1R consists of a heterodimer; this report suggests a similar mechanism for TLR1 and TLR2, for certain agonists. These data further suggest that mammalian responsiveness to some bacterial products may be mediated by combinations of TLRs, suggesting a mechanism for diversifying the repertoire of Toll-mediated responses.