Intestinal intraepithelial lymphocytes (IEL) are a population of cells consisting mostly of CD8+ T lymphocytes. Although their function is unknown, because of their location within the epithelium it has been postulated that IEL may be involved in defense against infection of the gut mucosa by pathogens including viruses. To address this issue, we have examined IEL populations from BALB/c mice systemically infected with lymphocytic choriomeningitis virus (LCMV). Viral infection induced a virus-specific cytotoxic response by IEL at 8 days postinfection. This virus-specific cytotoxic T lymphocyte (CTL) response was MHC class I restricted, and as is true for splenic T cells, recognition of viral antigen occurred predominantly in the context of the Ld molecule. The effector cells could be depleted by treatment with anti-CD8 antibody plus complement. In vivo treatment of mice with anti-alpha beta T cell receptor (TCR) antibody during the course of viral infection abrogated the response, suggesting that the virus-specific CTL were cells that express the alpha beta rather than gamma delta TCR. Consistent with this, no virus-specific IEL response could be detected in athymic mice, which have TCR gamma delta+ but not TCR alpha beta+ IEL. LCMV antigen could not be detected in the epithelium of the intestine, suggesting that viral antigen may have been encountered elsewhere. These data demonstrate for the first time a specific response by IEL to virus given by a non-oral route, and they suggest that thymus-derived alpha beta T cells can migrate to the intestinal epithelium following activation, where they may play a role in the response to virus and perhaps other infections.