Intraduodenal priming of mice with reovirus serotype 1/strain Lang (reovirus 1/L) stimulates gut lymphocytes and generates precursor and effector CTLs. Our earlier studies demonstrated that germinal center and T cell Ag (GCT) is a marker which identifies reovirus 1/L-specific precursor CTL and effector CTL in Peyer's patches (PP) of reovirus 1/L-inoculated mice. In this study, we characterized the expression of the activation markers, GCT and CD11c, on reovirus 1/L-stimulated gut lymphocytes and the effector mechanisms involved in reovirus 1/L-specific cytotoxicity. We found that intraduodenal reovirus 1/L inoculation of mice induced the expression of both GCT and CD11c on PP lymphocytes (PPL), intraepithelial lymphocytes (IEL), and lamina propria lymphocytes (LPL), and these activated cells expressed Fas ligand (FasL). The majority of the GCT+ CD11c+ IEL and LPL expressed a phenotype, TCRalphabeta+ Thy-1+ CD8+ similar to that expressed on reovirus 1/L-stimulated PPL. However, splenic lymphocytes expressed GCT but not CD11c after stimulation with reovirus 1/L. Perforin, Fas-FasL, and TRAIL pathways were found to be involved in PPL, IEL, and LPL cytotoxic activity against reovirus 1/L-infected targets. In PPL, perforin and Fas-FasL pathways were more effective than TRAIL. In IEL, all three cytotoxic mechanisms were equally as effective. However, LPL prefer Fas-FasL and TRAIL over perforin. Further, we demonstrated the preferential migration of GCT+ PPL to the intraepithelial compartment and the lamina propria. These results suggest that GCT and CD11c can be used as activation markers for gut lymphocytes and CD11c can also be used to differentiate between activated gut and systemic lymphocytes.