Chitosan, the deacetylated derivative of chitin, can be found in the cell wall of some fungi and is used in translational applications. We have shown that highly purified preparations of chitosan, but not chitin, activate the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome in primed mouse bone marrow-derived macrophages (BMMPhi), inducing a robust IL-1beta response. In this article, we further define specific cell types that are activated and delineate mechanisms of activation. BMMPhi differentiated to promote a classically activated (M1) phenotype released more IL-1beta in response to chitosan than intermediate or alternatively activated macrophages (M2). Chitosan, but not chitin, induced a robust IL-1beta response in mouse dendritic cells, peritoneal macrophages, and human PBMCs. Three mechanisms for NLRP3 inflammasome activation may contribute: K(+) efflux, reactive oxygen species, and lysosomal destabilization. The contributions of these mechanisms were tested using a K(+) efflux inhibitor, high extracellular potassium, a mitochondrial reactive oxygen species inhibitor, lysosomal acidification inhibitors, and a cathepsin B inhibitor. These studies revealed that each of these pathways participated in optimal NLRP3 inflammasome activation by chitosan. Finally, neither chitosan nor chitin stimulated significant release from unprimed BMMPhi of any of 22 cytokines and chemokines assayed. This study has the following conclusions: 1) chitosan, but not chitin, stimulates IL-1beta release from multiple murine and human cell types; 2) multiple nonredundant mechanisms appear to participate in inflammasome activation by chitosan; and 3) chitin and chitosan are relatively weak stimulators of inflammatory mediators from unprimed BMMPhi. These data have implications for understanding the nature of the immune response to microbes and biomaterials that contain chitin and chitosan.