Two-color FACS analysis of mouse bone marrow reveals a rare population, comprising 0.1-0.3% of the total, that expresses low levels of the Thy- 1 antigen but does not express any of five surface markers that characterize differentiated hematolymphoid cells. We demonstrate here that this fraction of mouse bone marrow is enormously enriched in cells that can home to the thymus and differentiate into mature T lymphocytes, subsequently migrating to peripheral lymphoid organs. Only a subset of the FACS-isolated fraction (1/90 after intrathymic injection) is capable of responding to the thymic microenvironment with a productive commitment to the T cell lineage. A second fraction of mouse bone marrow, which expresses how levels of Thy-1 but is also positive for at least one of five hematolymphoid lineage-specific markers, also contains cells that home to the thymus and establish colonies of thymocytes. The two fractions each contribute approximately equal amounts of thymic colony-forming units (CFUt) to the bone marrow, and together can account for at least half of the CFUt in whole bone marrow.