Recent studies in mice and humans have provided evidence for regional specialization of gut intraepithelial lymphocytes (IEL). Here the authors report striking regional variability in the composition of IEL in rat small and large intestine. Two-colour immunofluorescence in situ analysis showed that the distribution of the CD3+ and CD3- IEL subpopulations varied, the proportion of T cells (CD3+) being higher in the ileum than in the jejunum and smallest in the colon. These differences were explained by variable numbers of the T-cell receptor (TCR)alpha/beta + (both CD8+ and CD4+) but not the TCR gamma/delta + subset. Moreover, the various IEL subpopulations showed distinct intraepithelial distribution patterns with CD4+ and CD8 alpha beta + T cells situated near the lamina propria, while CD3- IEL were located preferentially towards the adluminal part of the epithelium. Regional phenotypic variation did not depend on intestinal colonization because analogous results were obtained in germ-free rats. Conventionalization nevertheless caused a marked relative increase of small intestinal TCR alpha/beta + but not TCR gamma/delta + IEL. This increase was more sustained in the jejunum than ileum and eventually reduced the phenotypic IEL differences between the two sites. By contrast, microbial colonization of the colon induced only a transient increase of intraepithelial TCR alpha/beta + cells with no permanent phenotypic alterations. Both CD3+ and CD3- IEL contained subpopulations that expressed NKR-P1 independent of intestinal colonization. These results demonstrate phenotypic specialization of IEL at different levels of the gut and suggest that the indigenous flora is not essential to this end.