The aim of this procedure is to obtain large numbers of isolated, viable, and functional mononuclear cells that are representative of the lymphoid population present in the mucosa of the human gastrointestinal tract under physiological and pathological conditions. The basic protocol is based on the use of surgically resected small and large bowel specimens, and consists of two basic stages: (1) a combination of chemical, enzymatic, and mechanical treatments to dissociate intestinal tissue and free the mononuclear cells from the surrounding interstitial framework; and (2) separation, isolation, and purification of viable mucosal lamina propria mononuclear cells from other cellular and amorphous components. The proportion of viable cells obtained can be increased by including extra separation steps using nylon wool columns or Percoll gradients, as described.