An accessory cell line, designated line A, was generated from bone marrow stem cells which differentiated in vitro in response to colony-stimulating factor (CSF) in substratum cultures. The cells were found to constitutively secrete large amounts of CSF, the activity of which was neutralized by anti-CSF-1 antibodies. Cells of line A and its supernatants potentiate the suboptimal response of thymocytes to PHA, manifesting an interleukin-1 (IL-1)-like activity. Culture fluids of this line also reconstitute the response to T cell mitogens of spleen cells depleted of adherent accessory cells. It was also found that cells of line A bear low levels of surface Ia, and they efficiently present soluble antigen to proliferating memory T cells. Constitutive prostaglandin secretion, which sometimes masks antigen-presenting capacity, was also demonstrated. Cells of line A are poorly phagocytic, do not secrete lysozyme, and lack Fc and complement receptors. However, they manifest strong cytoplasmic nonspecific esterase staining and an ectoenzyme profile resembling that of elicited inflammatory macrophages. In addition, the cell surface antigen Mac-2 was demonstrated, while stainings with anti-Mac-1 and anti-Mac-3 were negative. Thus, line A may represent a unique subpopulation of immunoregulatory accessory cells, the features of which are discussed.