We have investigated the origin of intraepithelial lymphocytes (IEL) populations in the murine gut, using reconstitution experiments in which the presence of thymus-derived cells of host or donor origin is rigorously controlled: RAG-/- mutant mice which have no T cells, were injected either with the bone marrow (BM) cells of nude mice or with selected peripheral lymph node (LN) T cells of euthymic mice. In thymectomized RAG-/- mice, injection of BM cells from nude mice led, after 2 mo, to the development of a peripheral B cell compartment and to the appearance, in the gut, of IEL bearing homodimeric CD8 alpha chains and either gamma/delta or alpha/beta TCR. In RAG-/- mice with a thymus, a similar injection led to complete lymphoid reconstitution, with the additional appearance in the gut of CD4+, CD8 alpha/beta+ or CD4+CD8 alpha/alpha+ IEL, all bearing alpha/beta TCR. In contrast, injection of LN T cells into these mice reconstituted a gut IEL population made of CD4+, CD8 alpha/beta+, or CD4+ CD8 alpha/alpha+ cells, all bearing alpha/beta TCR; CD8 alpha/alpha+ TCR-gamma/delta+ or alpha/beta+ IEL were not observed. These results demonstrate that the thymus and/or thymic-derived peripheral T cells are absolutely required for the generation of CD4+, CD8 alpha/beta+, and CD4+CD8 alpha/alpha+ IEL, which are thus thymus dependent. In contrast, TCR+ CD8 alpha/alpha+ IEL appear in the absence of the thymus, and thus are thymus independent.