Objectives: The aim of this study was to identify new markers of mucosal T cells to monitor ongoing intestinal immune responses in peripheral blood. Methods: Expression of cell-surface markers was studied in mice on ovalbumin (OVA)-specific T cells in the gut-draining mesenteric lymph nodes (MLN) after OVA feed. The effect of the local mucosal mediators retinoic acid (RA) and transforming growth factor-Β (TGF-Β) on the induction of a mucosal phenotype was determined in in vitro T-cell differentiation assays with murine and human T cells. Tetramer stainings were performed to study gluten-specific T cells in the circulation of patients with celiac disease, a chronic small-intestinal inflammation. Results: In mice, proliferating T cells in MLN were CD62Lneg CD38 during both tolerance induction and abrogation of intestinal homeostasis. This mucosal CD62Lneg CD38 T-cell phenotype was efficiently induced by RA and TGF-Β in mice, whereas for human CD4 T cells RA alone was sufficient. The CD4 CD62Lneg CD38 T-cell phenotype could be used to identify T cells with mucosal origin in human peripheral blood, as expression of the gut-homing chemokine receptor CCR9 and Β 7 integrin were highly enriched in this subset whereas expression of cutaneous leukocyte-associated antigen was almost absent. Tetramer staining revealed that gluten-specific T cells appearing in blood of treated celiac disease patients after oral gluten challenge were predominantly CD4 CD62Lneg CD38 . The total percentage of circulating CD62Lneg CD38 of CD4 T cells was not an indicator of intestinal inflammation as percentages did not differ between pediatric celiac disease patients, inflammatory bowel disease patients and respective controls. However, the phenotypic selection of mucosal T cells allowed cytokine profiling as upon restimulation of CD62Lneg CD38 cells interleukin-10 (IL-10) and interferon-γ (IFN-γ) transcripts were readily detected in circulating mucosal T cells. Conclusions: By selecting for CD62Lneg CD38 expression that comprises 5-10% of the cells within the total CD4 T-cell pool we are able to highly enrich for effector T cells with specificity for mucosal antigens. This is of pivotal importance for functional studies as this purification enhances the sensitivity of cytokine detection and cellular activation. © 2011 by the American College of Gastroenterology.