We describe a quantitative intrathymic (i.t.) adoptive transfer system for detecting thymocyte precursor cells in rats and mice. In this system, the generation of donor-origin thymocytes is analyzed on the FACS after the injection of test cells directly into the thymus of sublethally irradiated, histocompatible, RT-7 (rat) or Ly-1 (mouse) alloantigen-disparate recipients. Like the standard i.v. adoptive transfer assays for prothymocytes, the i.t. transfer assay is time, dose, and irradiation dependent. However, unlike the i.v. assays, the i.t. assay is highly sensitive, independent of cell migration, and specific for T-lineage precursor cells. Thus, the i.t. system requires between 25- and 50-fold fewer precursor cells than do the i.v. systems to generate a given number of donor-origin thymocytes; it detects nonmigratory as well as migratory subsets of precursor cells; it detects prethymic and intrathymic precursor cells with equal facility; and it produces a discrete, self-limited wave of donor-origin thymocytes and peripheral T cells. Moreover, neither hemopoietic nor lymphopoietic stem cell chimerism occurs at extrathymic sites. Comparison of the kinetics of thymocytopoiesis in the i.t. and i.v. transfer systems suggest that the seeding efficiency of prothymocytes in the i.v. assay approximates 0.04; the lag phase of the time-response curve is not due to a delay in the entry of prothymocytes into the thymus; and the relative amount of thymocyte precursor activity in various lymphohemopoietic tissues is highest in bone marrow, lowest (or absent) in lymph node, and intermediate in spleen, blood, and thymus. Moreover, the occurrence of saturation kinetics in the dose-response curve of the i.t. system supports the hypothesis that a finite number of microenvironmental niches for prothymocytes may exist in the thymus. These initial observations will require confirmation and extension in future studies. However, based on the present findings and related observations, we anticipate that the i.t. adoptive transfer system will contribute importantly to the definitive analysis of both normal and abnormal thymocytopoiesis.