Intestinal intra-epithelial lymphocytes (IELs) form a highly specialized lymphoid compartment. IELs consist primarily of T cells that are dispersed as single cells within the epithelial cell layer that surrounds the intestinal lumen. These lymphocytes along with lamina propria lymphocytes are considered to play an important role in the regulation of immune responses. IELs are heterogeneous with regard to phenotype, and they contain sub-populations with diverse functions. In our most recent study, we found that intra-duodenal inoculation of mice with reovirus serotype 1/strain Lang (reovirus 1/L) induced expression of both germinal center and T cell antigen and CD11c on IELs suggesting these cells to be the recently stimulated cells in gut mucosal tissue. We also demonstrated that IELs from these mice when cultured in vitro in the presence of reovirus 1/L-pulsed antigen-presenting cells generated reovirus 1/L-specific MHC-restricted CTL whose function was mediated utilizing perforin, Fas-FasL and TRAIL mechanisms. This present study provides a comprehensive analysis of the diverse subsets of IELs, which function with other mucosal cells to provide a strong, protective immunity in a highly regulated fashion inside the microenvironment of the intestinal epithelium. We demonstrated that the IEL population contains both thymus-dependent (TD) and thymus-independent (TI) lymphocytes in mice and that a complex phenotype is present when sub-populations are analyzed for TCR, Thy-1, CD4, CD8 and B220 expression in a comprehensive manner. In reovirus 1/L-inoculated mice, we found a decrease in the TI population and an increase in the TD population characterized by significant alterations in various sub-populations. This increase was largely due to an increase in CD4(+), CD8(+) and CD4/CD8 double-positive sub-populations of TD IELs. Intracellular cytokine analysis demonstrated induction of IFN-gamma and an increase in effector/cytotoxic CD8 and CD4 cells after reovirus 1/L infection. These results suggest that TD IELs may play an important role in the clearance of reovirus 1/L infection from gut.