Cell transfer experiments in congenic chick strains, one of which expresses the ov antigen marker, indicate that intestinal gamma delta T cells are derived from gamma delta+ thymocytes in embryos and newly hatched birds, and this early intestinal colonization occurs in two discrete waves. Here, we extend these studies to show that splenic colonization by gamma delta T cells occurs in essentially the same way. Following the engraftment of ov+ thymic lobes in thymectomized ov- recipients, gamma delta T cells migrate both to the spleen and intestine. By 1 week after hatching, a third generation of thymus-derived gamma delta T cells begins to migrate to both peripheral lymphoid organs, and this thymus-dependent seeding process is sustained over the first weeks of life. The survival time for splenic gamma delta migrants is significantly less than for the intestinal migrants. Tissue section analysis indicates that gamma delta T cells enter the intestinal epithelium at all villus levels. A shift in the gamma delta intraepithelial lymphocyte distribution toward the villus tip in thymectomized birds suggests the comigration of enterocytes and gamma delta intraepithelial lymphocytes. However, survival kinetics of the donor gamma delta population and a relatively high division rate of intestinal gamma delta T cells indicate that founder thymic migrants produce relatively long-lived clones of intestinal gamma delta T cells.