The intestinal mucosa represents a challenging environment for CD8+ T cells, which must tolerate nutrient antigens and commensal microorganisms while responding efficiently to pathogens. Consequently, specific regulatory mechanisms apply for CD8+ T cells in the intestinal environment, which should also be reflected in a tissue-specific gene expression profile of these cells. This study investigates whether such tissue-specific gene expression can be observed in CD8+ T cells primed during bacterial infection. To identify intestine-specific gene expression in conventional CD8alphabeta+ T cells, mice were infected with Listeria monocytogenes expressing ovalbumin (LmOVA). Using OVA257-264 tetramers, specific CD8+ T cells were sorted from spleen, liver and the small intestinal mucosa, and RNA samples from these cells were compared using microarrays. This approach allowed the identification of differences in gene expression in a highly defined CD8+ T-cell population with identical antigen specificity generated during infection. One group of genes with reduced expression in the intestinal mucosa comprised members of the C-type lectin-like natural killer receptor (NKR) family. Fluorescence-activated cell sorting analysis was used to assess protein expression of NKR. NKR expression on CD8+ T cells from the intestinal mucosa was dependent on the route of listeria application and consequently on the site of T-cell priming. Retinoic acid influenced NKR expression consistent with an imprinting of the NKR expression profile in intestine-associated lymphoid tissues. In contrast, NKR expression was largely independent from intestinal flora. Our results demonstrate that in the intestinal mucosa, conventional CD8alphabeta+ T cells lack NKR expression and thereby lose responsiveness to NKR ligands, which otherwise could possibly cause adverse activation or inhibition of T cells in this environment.