Recognition of potentially pathogenic organisms is a critical first step in an immune response. To prevent pathogen outgrowth, this detection must be rapid and initiate a robust inflammatory response. However, inflammation is dangerous, and spurious activation in response to nonthreatening stimuli has the potential to cause autoimmunity and other inflammatory disorders. For this reason, the pattern recognition receptors (PRRs) of the innate immune system have evolved to recognise pathogen-associated molecular patterns to distinguish between self and potentially infectious nonself. The first discovered and most well-studied PRRs are the toll-like receptors (TLRs), which are transmembrane receptors that detect a diverse set of microbe-specific products. Extensive work has uncovered the proteins required in TLR signalling, but a more complete understanding of the biochemistry of signalling molecules in their cellular context is required to understand the role of TLRs in pathogen detection and clearance.