Analysis of kinetics of thymic repopulation by Rh123low, Lin-, Ly6A/E+, c-kit+ (Rh123low) cells, highly enriched for long-term in vivo hematopoietic repopulating cells, reveals that this population is deficient in thymic repopulation at week 3 after intravenous transplantation when compared to normal bone marrow cells. This suggests that the marrow prothymocytes have been depleted from this population, and analysis of thymic repopulation at week 3 can therefore be used to differentiate prothymocytes and their precursors. Using this short-term assay, the Rh123high, Lin-, Ly6A/E+, c-kit+ (Rh123high) population has been found to be relatively more efficient at early thymic repopulation, suggesting that this population contains the prothymocytes. In addition, the differentiation potential and reconstitution behavior of the Rh123high population observed after intravenous and intrathymic transfer strongly indicates that this population is at the transitional stage between the marrow primitive pluripotential and thymic more mature lymphoid restricted stem cells. We propose that the thymic repopulating ability of the Rh123low population is through generation of the more mature Rh123high progeny, presumably in the marrow.