Macrophages respond to Salmonella typhimurium infection via Ipaf, a NACHT-leucine-rich repeat family member that activates caspase-1 and secretion of interleukin 1beta. However, the specific microbial salmonella-derived agonist responsible for activating Ipaf is unknown. We show here that cytosolic bacterial flagellin activated caspase-1 through Ipaf but was independent of Toll-like receptor 5, a known flagellin sensor. Stimulation of the Ipaf pathway in macrophages after infection required a functional salmonella pathogenicity island 1 type III secretion system but not the flagellar type III secretion system; furthermore, Ipaf activation could be recapitulated by the introduction of purified flagellin directly into the cytoplasm. These observations raise the possibility that the salmonella pathogenicity island 1 type III secretion system cannot completely exclude 'promiscuous' secretion of flagellin and that the host capitalizes on this 'error' by activating a potent host-defense pathway.