T cell precursors located in peripheral immune tissues have been studied according to the potential to repopulate the thymus and the gut of lethally irradiated mice. T cell repopulation could be achieved with spleen cells from athymic or euthymic mice thoroughly devoid of mature T cells. Repopulation did not occur with lymph node lymphocytes as determined from studies in congenic mice. The kinetics of T cell repopulation differed in the gut and thymus in that donor-derived T cells appeared in the gut by day 7 after cell transfer, and in the thymus by day 14 after cell transfer. The multipotent nature of splenic T cell precursors was evident from the finding that all major phenotypic subsets of T cells in the thymus and the gut developed after spleen cell transfer. T cell repopulation of the intraepithelial lymphocytes also occurred efficiently in athymic radiation chimeras injected with spleen cells from congenitally athymic nude mice, demonstrating that gut T cell repopulation by those cells does not require a functional thymus. PCR-spectratype analyses of twenty-four V beta TCR genes in thymocytes and intraepithelial lymphocytes revealed extensive TCR-beta repertoires in both tissues 1 to 2 wk after cell transfer, although T cells with rearranged TCR were undetectable in the donor spleen cell population. The minimal phenotype of the splenic T cell precursor was determined to be CD3-, CD4-, CD8-, HSA+.