Stromal cells which grow as an adherent layer of Whitlock-Witte cultures are thought to be an essential component of the lymphohemopoietic microenvironment. Stromal cell lines from bone marrow (BM) and spleen have been obtained by treatment of cultures with 5-fluorouracil and selected for their lymphocyte support capacity by measuring the clonal growth of stromal cell-dependent lymphocyte lines in methyl cellulose. Established stromal cell lines differed significantly from stromal cells in primary Whitlock-Witte cultures with respect to expression of certain hemopoietic cell surface markers. For example, the Thy-1 and Mac-3 antigens were expressed by stromal cell lines obtained from BM and spleen, but not by stromal cells in primary cultures. Features common to all stromal cells include synthesis of actins, the neural adhesion molecule N-CAM, and a variety of collagens. Two types of common leukocyte antigens were not significantly expressed. The proliferation and total protein synthetic capacity of lymphocyte-supportive stromal cell lines was sensitive to ionizing radiation. After exposure of the cells to 200 rads, the incorporation of either [3H]thymidine or [3H]Leucine was reduced to less than 50% of control values, but the growth of lymphocytes was augmented in the presence of an irradiated stromal cell layer. The proliferation of stromal cell lines was also affected by exposure to a variety of growth factors. Addition of epidermal growth factor or endothelial cell growth factor augmented BM or spleen-derived stromal cell proliferation, while interferon-gamma had the opposite effect. In general, but not exclusively, lymphocyte growth was inhibited by factors which augmented the proliferation of stromal cells. Novel methods are described for isolating stromal cells and determining their capacity to support lymphocyte growth in vitro. Evidence is presented that this ability is not restricted to BM-derived stromal cells. The function of stromal cells was not dependent on their ability to proliferate, and this may be modulated by immunoregulatory and other growth factors.