Populations of lymphoid cells from the gastrointestinal tract of guinea pigs were compared with splenic cells for both morphologic and functional characteristics. Gastrointestinal lymphoid cells were isolated from mesenteric lymph nodes, Peyer's patches, and also for the epithelium and lamina propria of the small intestinal mucosa. Isolated intraepithelial and lamina propria lymphocyte populations contained (a) T cells, B cells, and macrophages; (b) T cells that proliferated to phytohemagglutinin and concanavalin A; and (c) B cells that proliferated to bacterial lipopolysaccharide. Using inbred guinea pigs, the intraepithelial and lamina propria lymphocytes were shown to contain T cells capable of proliferating to alloantigens in mixed leukocyte culture, and also cells capable of stimulating alloreactive T cells. These studies demonstrate the presence of immunologically reactive T cells and B cells in isolated small intestinal mucosal lymphoid cells from guinea pigs.