Because murine intestine intraepithelial lymphocytes (IEL) are dispersed throughout the intestine epithelium, it is important that IEL extraction procedures result in lymphocyte preparations of sufficient purity for use in in vitro and in vivo experimental systems. Here, we describe an improved technique for isolating murine IEL consisting of a single 30 min extraction followed by multiple nylon wool filtrations and centrifugation through Percoll. This procedure yields a preparation of IEL with high overall recovery and purity yet takes only 2-2.5 h. Evaluation of individual steps in the extraction process indicated that nylon wool filtration, in particular multiple filtrations, and Percoll fractionation both were important for achieving highly-enriched IEL populations by removal of enterocytes and cellular debris, and demonstrated that multiple nylon wool filtration improved the overall IEL recovery. This procedure has several advantages for studies of murine IEL in that the resultant IEL population is ideal for phenotypic, functional, or molecular analyses. Moreover, this technique is effective for isolating IEL on a single-animal basis, thereby permitting analyses of IEL from individual mice rather than as pooled IEL obtained from several animals.