We explored the role that thrombospondin (TSP), a multifunctional extracellular matrix protein, plays in hematopoietic cell-cell and cell- matrix interactions. Thrombospondin synthesis is differentially regulated in human long-term bone marrow cultures. Consistent with this, human hematopoietic progenitor cells of all three lineages (erythrocyte, megakaryocyte, and granulocyte) use TSP as an attachment protein. However, terminally differentiated cells (erythrocytes and neutrophils) show absent or reduced attachment to TSP. The region within the TSP molecule that mediates cell attachment (cell binding domain) was delineated by examining both attachment to proteolytic fragments of TSP and by inhibition of cytoadhesion using monoclonal antibodies directed against TSP domains. The cell binding domain resides toward the C-terminus of a 140 Kd chymotryptic fragment of TSP. We conclude that thrombospondin functions as a hematopoietic cytoadhesion molecule, capable of binding primary hematopoietic progenitor cells, and may, therefore, be important in blood cell development.