It has been proposed that intestinal intraepithelial lymphocytes (I-IEL) perform immune surveillance of the epithelial layer (1) and regulate mucosal humoral responses to exogenous Ag (2). To better understand the functional potential of this unique population, purified murine I-IEL were analyzed phenotypically and functionally. Initial studies determined that I-IEL could be distinguished based on several phenotypic characteristics including: TCR (TCR-alpha beta vs TCR-gamma delta); Thy-1, CD45R/B220, CD5, and CD8 (CD8 alpha alpha vs CD8 alpha beta) expression. Using anti-TCR mAb, individual I-IEL subsets were activated and examined functionally. Both TCR-alpha beta and TCR-gamma delta I-IEL were found to synthesize an array of lymphokines that included IL-2, IL-3, and IL-6 but not IL-4 or IL-5. Additionally, a number of lymphokines were detected that directly influence epithelial function (IFN-gamma, TNF-alpha, and TGF-beta 1). However, the majority of the I-IEL function was localized within the Thy-1+, CD45R/B220- I-IEL subset. In addition those TCR-alpha beta I-IEL expressing the CD8 alpha beta heterodimer were more easily activated. Thus, a subset of I-IEL have the capacity to respond to TCR-mediated stimuli. The functional activities of these cells may influence both local immune cell populations as well as epithelial differentiation.