The lymphocytes of epithelial and lamina proprial compartments of the intestine are phenotypically and functionally distinct and serve a wide range of functions in the intestinal mucosa like regulating intestinal homeostasis, maintaining epithelial barrier function as well as regulating adaptive and innate immune responses. To analyze the role of these cells in different disease states, it is necessary to isolate pure cell populations of the intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) of the gut. In this protocol we describe a method to isolate T cells from IEL and LPL, which can be used for further investigations like comparative studies of mRNA expression, cell proliferation assay, or protein analysis.