Intraepithelial intestinal T lymphocytes (IEL) bearing alpha beta or gamma delta T cell receptors (TCR) are positioned to serve as a first line of defense against enteric pathogens. To investigate whether intestinal IEL are subject to antigenic selective forces distinct from that influencing (xp T cells in the peripheral blood (PBL), we performed a comparative analysis of V beta gene segment usage in IEL and PBL of immunologically normal donors by quantitative PCR. Primers for 22 different human TCR V beta gene segments of V beta gene segments families were used to analyze the repertoire of TCR beta chain transcripts in colonic IEL (c-IEL), in corresponding colonic lamina propria lymphocytes (c-LPL), and in peripheral blood lymphocytes. In each of the three individuals examined, a limited number (1-4 out of 22) of TCR V beta families predominated and accounted for more than 50% of the total beta chain transcripts from c-IEL, whereas in PBL and c-LPL a more even distribution of V beta gene families was observed. The dominating V beta gene families were V beta 2, V beta 3, V beta 6, V beta 8 and V beta 14. In one individual, V beta 3 comprised more than 40% of the entire repertoire of c-IEL beta chain transcripts. Sequence analysis of the predominant V beta 3 family in this individual revealed identical sequences in 13 of 17 clones analyzed. Human alpha beta TCR+ c-IEL could not be driven to proliferate or exhibit cytotoxic function in vitro however, PCR analysis for detection of lymphokine mRNA revealed constitutive production of several lymphokines known to exert trophic effects on intestinal epithelial cells and pro-inflammatory activities. Taken together, the striking degree of oligoclonality may indicate that the intraepithelial intestinal immune system is targeted to a limited set of hitherto unknown self- or foreign antigens present in the intestine and orchestrates intramucosal inflammatory and regenerative processes.