The study of intestinal intraepithelial γδ lymphocytes has been hindered by the difficulty of isolating a population of pure cells representative of their in vivo phenotype and function. Here, we describe a procedure for the purification of small intestinal intraepithelial γδ T lymphocytes in BALB/c mice, including extraction and purification of intraepithelial lymphocytes (IELs) followed by separation of γδ T cells from IELs by magnetic activated cell sorting (MACS). The small intestinal IEL isolation protocol reported here is a less time-consuming with high purity and high yields. One-step Percoll purification of IELs using a 40%-70% discontinuous Percoll gradient is less laborious yet sufficient to get exact profiles during phenotypic analysis of cell subsets. IELs obtained after two-step Percoll centrifugation using 30% Percoll, followed by a 40%-70% discontinuous Percoll gradient, are fit to purify γδ T cells by MACS. Small intestinal IELs produce much more IFN-γ and TGF-β1 than sorted γδ T cells upon stimulation with plate-bound anti-CD3ε mAb. γδ T cells secret moderate amounts of TGF-β1 and minimal amounts of IFN-γ. Purification of γδ T cells is helpful in investigating the phenotype and function of intestinal mucosal γδ T cells.