The murine clonal preadipose cell line, MC3T3-G2/PA6 (PA6), has the ability to support in vitro proliferation of hematopoietic stem cells defined as colony-forming units in spleen (CFU-S). In order to ascertain the relationship between the hematopoietic-supporting activity of PA6 cells and their expression, we cultured a number of these cells for over 45 weeks and investigated the level at which they expressed several cell surface markers and membrane-bound growth factors. Besides expressing stem cell factor (SCF) and macrophage colony-stimulating factor (M-CSF), PA6 cells were found by flow cytometry analysis to express high levels of stem cell antigen-1 (Sca-1). The expression level of Sca-1 in PA6 cells correlated with the ability of the latter to support hematopoiesis, whereas no such correlation was observed in the case of SCF and M-CSF expression. A cDNA clone encoding the protein recognized by anti-Sca-1 antibody was isolated from PA6 cells by expression cloning, so that its nucleotide sequence encoded the protein identical to mouse alloantigen Ly-6A.2. Genetically engineered COS-7 cells, transformed by the expression vector carrying the Ly-6A.2 gene, suppressed proliferation of murine lineage marker-negative (Lin) bone marrow cells by themselves and synergistically augmented proliferation of these cells in the presence of SCF. These results suggest that Ly-6A.2 regulates the proliferation of hematopoietic progenitor cells, and is one of the molecules organizing the hematopoietic microenvironment provided by stromal cells.