Cystic fibrosis (CF) patients suffer recurrent pulmonary infections that are characterized by an overactive, yet ineffective and destructive inflammatory response that is associated with respiratory infections by Pseudomonas aeruginosa, a pathogen which produces a number of phlogistic molecules. To better understand this process, we have utilized exoenzyme S (ExoS), one of the key Pseudomonas aeruginosa secreted exoproducts, which is known to stimulate cells via the Toll-like receptor (TLR) pathway. We found that ExoS induced pro-inflammatory cytokine production via the NF-KB, Erk1/2 and Src kinase pathways. Since Src kinases are concentrated within cholesterol-containing detergent resistant membrane microdomains (DRM), sometimes also called lipid rafts, and DRM act as signaling platforms and amplifiers on the surface of cells, we addressed the role of DRM in ExoS signaling. ExoS bound directly to a subset of DRM, and induced the phosphorylation of multiple proteins within DRM including Src kinases. Disruption of DRM by cholesterol extraction prevented NF-KB and Erk 1/2 activation, and TNF- production, in response to ExoS. Activation of monocytic cells by other TLR and Nod-like receptor agonists such as lipoteichoic acid, lipopolysaccharide and peptidoglycan were also dependent on DRM, and disruption prevented TNF- production. Disruption of DRM did not prevent ExoS binding, but did release the Src kinase, Lyn, from the DRM fraction into the detergent-soluble fraction, a site in which Src kinases are not active. These studies show that ExoS, a TLR agonist, requires direct binding to DRM for optimal signaling, which suggests that DRM are possible therapeutic targets in CF.