Human intraepithelial lymphocytes (IEL) are predominantly CD45RO+ (memory) CD8+ T lymphocytes located between intestinal epithelial cells. This study determines whether IEL share other characteristics with circulating CD45RO+ lymphocytes. Memory cells are large and have an upregulated expression of adhesion molecules. In contrast, the majority of IEL are the same size as peripheral blood (PB) T cells. In addition, IEL do not have an increased density of adhesion molecules, although a larger percentage express CD54 and CD58 compared to PBL. Allo-CTL activity, demonstrated by CD45RO+ PBL, could not be shown using either IEL or lamina propria lymphocytes (LPL) after a 5-day culture with alloantigen. However, after a 2.5-week culture with allogeneic PBL and interleukin-2 (IL-2), both IEL and LPL were capable of this cytotoxic function. The addition of IL-4 or IL-6 did not alter CTL activity by mucosal lymphocytes. Alloantigen-stimulated IEL, LPL, and PB CD8+ T cell lines were propagated for up to 16 weeks. All lines demonstrated alloantigen-specific proliferation. The PB CD8+ T lymphocytes maintained their phenotype and allo-CTL activity. In contrast, the CD4+ subset in the IEL and LPL became the predominant lymphocyte type and demonstrated potent lytic activity that was not alloantigen specific. This study shows that (1) IEL do not have increased density of adhesion molecules, (2) mucosal lymphocytes demonstrate allo-CTL activity, (3) CD8+ T cells in the mucosa cannot be perpetuated long-term by alloantigen and IL-2, and (4) mucosal CD4+ T cell lines demonstrate marked alloantigen-nonspecific cytotoxicity. The only characteristic shared by IEL and circulating memory cells is allo-CTL activity.