It is unclear whether human intestinal intraepithelial T lymphocytes (iIEL) are resting or activated cells. To address this question, an improved isolation procedure was developed for small bowel iIEL, which were analysed by two-colour flow cytometry and compared with resting and mitogen-activated peripheral blood lymphocytes, iIEL expression of CD44 isoforms, Bcl-2 and Ki67 antigen was also determined in tissue sections, iIEL expressed CD69 at levels comparable with 48-72 h phytohaemagglutinin blasts, but did not express CD25 or CD95. iIEL were Bcl-2+ but not Ki67+. αEβ7 and α4B7 expression was relatively high, whereas αLβ2, CD5 and CD28 were expressed at low density. Isolated iIEL expressed CD44 (core epitopes) at lower levels than peripheral blood lymphocytes, although almost all CD44 contained splice variant 6 (CD44v6). Peripheral blood lymphocytes expressed CD44 at very high density, but little CD44v6, even after activation. However, in tissue sections, iIEL showed differential labelling with CD44 core epitope antibodies and no detectable CD44v6, implying CD44 receptor occupancy or epitope masking in situ. Thus, normal iIEL express a quasi-activated phenotype with unusual patterns of adhesion receptors, which may act as costimulatory elements. These may permit iIEL to assume effector functions, with absence of CD25 preventing entry into the cell cycle, thereby maintaining an apoptosis-resistant phenotype.