Intraepithelial lymphocytes (IELs) have been extensively studied in the murine small intestine. However, to date no studies have assessed IEL in the large intestine, despite the marked differences in function and lumenal environment. In the present study, we isolated IEL from both small and large intestine of three mouse strains (BALB/c, C3H/HeN, C57BL/6) and determined the frequency of CD2, CD4, and CD8 expression on CD3+ IEL, as well as the frequency of αβ and γδ TCR usage and Vβ distribution. Higher numbers of IEL/unit length were always isolated from the small intestine (20-30 x 106/5 mice) compared with large intestine (1.1-2.5 x 106/5 mice). Interestingly, IEL from the large intestine of all strains were predominantly αβ TCR+ whereas γδ TCR+ IELs predominated in small intestine. Large intestinal IELs were mainly CD4+, in both BALB/c and C3H/HeN mouse strains. IELs from large intestine of C57BL/6 mice were mainly CD8+; however, the CD4+ subset was fourfold higher when compared with small intestine IEL. Potential functional differences between IEL subsets was assessed by determining the relative levels of mRNA for IL-1, 2, 4, 5, 10, IFN-γ, TGF-β, and TNF-γ. Similar patterns for IL-1, IFN-γ and TNF-α were seen while more IL-2, IL- 4, IL-5, and IL-10 mRNA was noted in large intestinal IEL. Stimulation of C3H/HeJ IEL with anti-CD3 also resulted in higher levels of IL-3/GM-CSF, IL- 4, and IL-6 by IEL from large intestine. These results show that marked differences occur among the T cell subsets present in IELs from mouse small and large intestine.