Successful long-term culture of murine thymic macrophages was achieved by plating adherent thymic cells, in the presence of L cell-conditioned medium, on dishes coated with an extracellular matrix. Adherent thymic cells in normal conditions of in-vitro culture do not proliferate. Those maintained on plastic tissue-culture dishes, and exposed to L cell-conditioned medium, proliferate slowly to a limited degree and form very small colonies. In contrast, when cultured in dishes coated with an extracellular matrix formed by corneal endothelial cells, in the presence of L cell-conditioned medium, adherent thymic cells proliferate rapidly and after 12-21 days in culture form large colonies (about 3-5 mm in diameter). The proliferating cells were identified to be mononuclear phagocytes by their morphological appearance, their ability to ingest both bacteria and antibody-coated erythrocytes and by their nonspecific esterase activity. These cells were also shown to exhibit cell surface antigens that are characteristic of differentiated macrophages, e.g. Fc receptors and the specific macrophage cell surface marker F4/80. A high percentage of these cultured cells were found to bear I-A antigens. The adherent thymic mononuclear phagocytes could be trypsinized and passaged while maintaining both their ability to proliferate and their specific macrophage characteristics for a period of 70 days. Thus, monocyte-macrophage stem cells ae present in the thymus, and under appropriate in-vitro conditions, can be made to proliferate and mature to I-A-bearing macrophages.