The intestinal mucosal immune system is composed of three major lymphoid areas: the lamina propria (LP), which lies just underneath the basement membrane in the intestinal villi; (2) the intraepithelial compartment, which contains the intraepithelial lymphocytes (IEL) and is located just above the basement membrane, between the columnar epithelial cells; and (3) Peyer's patches (PP), lymphoid nodules (akin to lymph nodes) embedded in the gut wall, separated from the LP and IEL. The LP, PP, and IEL lymphoid populations form a complex, interconnected network that responds to immunological insults in the intestine. Therefore, these lymphocyte populations should be analyzed when studying the immunological status of the intestine, for example in oral immunization or in intestinal disease (including infectious disease and tumors). This unit details techniques for isolation of IEL, PP cells, and LP cells from the small intestine of the mouse.