We have previously shown that the most primitive human hematopoietic cells are included within a cell subpopulation expressing high levels of CD34 and low or undetectable levels of CD45RA and CD71. In this study, cord blood cells with this phenotype were sorted and further separated based on their expression on the Thy-1 antigen. The proliferation and differentiation of the purified cell fractions in response to a mixture of hematopoietic cytokines was analyzed in serum- and stroma-free liquid cultures. Thy-1+ cells (25% of CD34+ CD45RAlo CD71lo cells) were particularly enriched for high proliferative potential colony-forming cells (HPP-CFC; up to 45% of the clonogenic cells), whereas Thy-1- cells were enriched for multipotential colony-forming cells (CFU-MIX; up to 46% of the clonogenic cells). When both subpopulations were cultured in serum-free liquid cultures supplemented with a cytokine mixture that included steel factor, interleukin-6 (IL-6), granulocyte-macrophage colony-stimulating factor (GM-CSF)/IL-3 fusion protein, M-CSF, G-CSF, and erythropoietin, Thy-1+ cells showed a much higher numerical expansion of CD34+ cells (30,000-fold) and colony-forming cells (4,700-fold) than was observed in cultures initiated with Thy-1- cells (900-fold increase in CD34+ cell numbers and 241-fold increase in CFC numbers). Cells coexpressing CD34 and Thy-1 were only transiently expanded (up to 29-fold) and were not detected after day 22 of culture. When CD34+ CD45RAlo CD71lo Thy-1+ cells were cultured, either in semi-solid or liquid cultures, in the presence of anti-Thy-1 antibody, a significant reduction in progenitor cell numbers (particularly HPP-CFC) was observed. In contrast, CD34+ CD45RAlo CD71lo Thy-1- cells were not affected by anti-Thy-1. The results of this study indicate that Thy-1 is expressed on primitive cord blood progenitors with the highest in vitro proliferative potential, and further suggest that Thy-1 is involved in hematopoietic cell development, possibly by mediating a negative signal that results in inhibition of primitive cell proliferation.