It is well established that CD7, gp40 antigen is one of the first antigens detected on the surfaces of cells of the human T-cell lineage. Using complement-dependent cytotoxicity and immunoadherence to anti-CD7-coated surfaces, we were able to purify CD7+2-3-4-8-TcR- cells with greater than 90% purity from both human thymus and bone marrow. Limiting dilution analysis showed that these cells displayed high ability to generate mature T-cell clones when they were cultured in the appropriate conditions. These precursors needed phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM) as a differentiation signal before being able to respond to PHA and recombinant interleukin 2 (rIL2). CD7+CD2- precursors differed from more mature CD7+CD2+ thymocytes because they were not sensitive to PHA, IL2, or CD2 triggering. Bone marrow-derived clones were mostly CD4+, whereas thymic cells generated more CD8+ than CD4+ clones. Together, this study indicates that the CD7+CD2- precursor is one of the earliest prothymocytes able to differentiate and proliferate in vitro.