Stromal cell lines were established by irradiating adherent layers of bone marrow and spleen cells in Dexter-type long-term culture with X-rays. Some of these cell lines support myelopoiesis and/or B lymphopoiesis in vitro. Furthermore, the characteristics of these stromal cell lines were studied. Cytokine activity was detected in the conditioned media from all hematopoietic-supportive and non-supportive stromal cells. Quantitative reverse transcriptase-polymerase chain reaction analysis revealed that the mRNAs of macrophage colony-stimulating factor and stem cell factor, but not that of Interleukin-3, were detectable in all the hematopoietic-supportive and non-supportive stromal cell lines. The transcripts of granulocyte colony-stimulating factor, interleukin-6, interleukin-7, and leukemia inhibitory factor were expressed in a wide variety of cell lines. Most stromal cell lines synthesized mRNA of c-mpl, the ligand of which stimulates megakaryopoiesis and thrombopoiesis. These observations indicate that the pattern of mRNA expression of cytokines is not correlated with the hematopoietic-supportive ability of stromal cell lines. There was a significant difference in the efficiency of adhesion of lineage marker-negative bone marrow cells to fibroblasts and stromal cell lines. This appears to be correlated with the hematopoietic-supportive ability of the stromal cell lines.