Located on the inside of the throat, the paired palatine tonsils form part of the first major barrier protecting the digestive and respiratory tracts from potentially invading microorganisms. The tonsils have a surface of stratified squamous epithelium that extends into deep and branched crypts lined by reticulated epithelium, which in parts may only be one cell thick. Organized in the sub-epithelial space are B cell rich lymphoid follicles. T cells are mostly located in the extra-follicular spaces with a very high CD4:CD8 T cell ratio. In addition to the T and B cell subsets, six phenotypes of dendritic cells (DC) have been identified in the tonsils: Langerhans cells in the squamous epithelium, germinal center DC, and follicular DC in the germinal center, and another three DC phenotypes that are located in the extra-follicular area (interdigitating DC, plasmacytoid DC, and lympho-epithelial symbiosis-DC). Here, we describe the isolation of tonsil mononuclear cells from fresh human tonsil.