BACKGROUND: The characteristics and roles of gut lymphocytes have been only partly elucidated, in particular with regard to activation patterns. OBJECTIVES: To characterize lymphocytes from various parts of the gut and examine their activation pattern as a network. METHODS: Lymphocytes were isolated from the epithelium, the lamina propria, Peyer's patches, mesenteric lymph nodes, the spleen, and peripheral blood of naive mice. They were then characterized for T cell phenotype, T cell receptors (TcRs), activation markers, and cytokine production. RESULTS: The results showed a gradient of cells with an increasing proportion of TcRgammadelta+, CD8alphaalpha+ cells towards the gut lumen, with the highest number found in intraepithelial lymphocytes. These cells, together with lamina propria lymphocytes (LPLs) were also characterized by a memory-like phenotype (CD25- CD45RB(low) and CD44(high)) and CD69 expression. CD8+ TcRgammadelta+ LPLs produced IL-10 and IL-17, while TcRalphabeta+ LPLs were FoxP3 positive. CONCLUSIONS: Gut lymphocytes express various receptors and cytokines according to their location. These specific features suggest a differential function for gut lymphocytes depending on their location.